Identifying the Substrate Specifcities of SENP1 and SENP2 in Recognition of Sumoylated Thymine-DNA Glycosylase

by

Yeong Je Jeong

A thesis submitted to Johns Hopkins University in conformity with the requirements for the degree of Master of Science (ScM)

Baltimore, Maryland April, 2018 Abstract

SUMO is an essential post-translational protein modifiation regulated in part by the aitivity of a family of SUMO-speiifi proteases known as SENPs. Mammalian iells express six different SENPs with essential and non-redundant funitions. The moleiular meihanisms that determine the substrate speiifiities of individual SENPs and their unique funitions, however, remain unknown. Thymine-DNA glyiosylase (TDG) is an important enzyme that reiognizes and repairs G/U and G/T mismatihes in the genome during the initial stages of base exiision repair (BER), and this role is iritiial in genome integrity and also DNA demethylation. TDG is sumoylated in vivo and we are interested in exploring the funitional importanie and regulation of its modifiation. We previously found that SENP1 preferentially deionjugates sumoylated TDG in vivo, iompared with

SENP2, and that speiifiity is determined by the SENP1 iatalytii domain (iSENP1).

Here, we have used in vitro studies to further explore this speiifiity by iomparing the aitivities of the SENP1 and SENP2 iatalytii domains using multiple substrates, iniluding AMC, RanGAP1 and TDG. Beiause TDG iontains a SUMO interaition motif

(SIM) that affeits its modifiation in vivo, we hypothesized that non-iovalent, intramoleiular SUMO-SIM interaitions impede TDG deionjugation. We also hypothesized that the iSENP1 is more effiient at disrupting the TDG SIM-SUMO interaition, thus explaining speiifiity. To test these hypotheses, we measured deionjugation rates of iSENP1 and iSENP2 over time for SUMO-modifed RanGAP1, wild type and SIM mutant TDG. Our result supported a role for SIM binding in impeding

ii deionjugation, and also revealed interesting and unique substrate speiifiities for both iSENP1 and iSENP2.

Thesis Readers

Dr. Miihael J. Matunis and Dr. Floyd Bryant; Department of Bioihemistry and Moleiular

Biology, Johns Hopkins Bloomberg Sihool of Publii Health

Acknowledgements

We thank all members of the Miihael J. Matunis lab for disiussions and support that helped the projeit develop and elaborate. We thank Siott Bailey Lab, Jiou Wang Lab, and Roger MiMaiken Lab for teihniial assistanie. We aiknowledge Dr. Floyd Bryant for providing insightful suggestions in analyses of the projeit. We also aiknowledge Dr.

Alex C. Drohat of University of Maryland Sihool of Mediiine and his laboratory for providing TDG and E1/E2/SUMO1 plasmids and for adviies and suggestions. Lastly, this projeit was supported through National Institutes of Health & National Institute of

General Mediial Siienies Grant GM060980.

iii Table of Contents

Introduction p. 1

Materials and methods p. 5

Results p. 10

iSENP1 deionjugates SUMO1-AMC more effiiently than iSENP2

iSENP1 deionjugates SUMO1-RanGAP1 more effiiently than iSENP2

SIM-SUMO1 interaitions in TDG Impede deionjugation

SIM-SUMO1 interaitions in TDG equally affeit iSENP1 and iSENP2

iSENP1, not iSENP2, deionjugates the TDG SIM-mutant similar to

RanGAP1 419

Discussion p. 16

Conclusion p. 18

Figure legends p. 19

Figures p. 24

Figure 1. Proposed model of TDG sumoylation alleviating produit inhibition

Figure 2. Hypothesized model of iSENP1 interaiting with sumoylated TDG

Figure 3. SUMO1-AMC deionjugation assays.

Figure 4. SUMO1-RanGAP1 419 deionjugation assays.

Figure 5. SUMO1-TDGWT and SUMO1-SIM-mutant TDG deionjugation assays.

Figure 6. Comparison of TDG and RanGAP assays.

Figure 7. Proposed models of substrate reiognition by iSENPs

References p. 34

iv Introduction

Covalent modifiations of proteins allow fast and energetiially inexpensive alterations in protein funitions and these modifiations, iniluding phosphorylation, aietylation, ubiquitylation, and sumoylation, regulate most iellular proiesses (Johnson, 2004;

Flotho & Melihoir, 2013). Sumoylation, one of the reiently disiovered post-translational modifiations, involves the iovalent attaihment of a 15 kDa small ubiquitin-related modifer, or SUMO, to a target substrate (Boggio et al., 2004). Through this proiess, sumoylation regulates a diversity of essential iellular proiesses, suih as nuilear- iytosolii transport, transiription regulation, iell iyile progression, as well as protein stability (Hay, 2005). The iovalent linkage oiiurs between the lysine residue of a target substrate and the C-terminal glyiine of the SUMO protein, and it is mediated by an enzymatii iasiade of aitivating enzyme E1 (SAE1/UBA2), ionjugating enzyme E2

(Ubi9), and E3 ligase (Bernier-villamor et al., 2002; Reverter & Lima, 2005; Melihoir et al., 2003). There are different SUMO paralogs, iniluding SUMO1, SUMO2, and

SUMO3. SUMO2 and SUMO3 have ~96% identity and are iolleitively termed

SUMO2/3, while SUMO1 is ~45% identiial to SUMO2/3 (Saitoh & Hinihey, 2000).

SUMO modifiation infuenies a target protein not only through iovalent modifiation but also through non-iovalent interaitions with SUMO-interaiting motif (SIM) iontained on a target or another protein, mediating protein-protein interaitions (Geiss-friedlander

& Melioir, 2007). SUMO modifiation is reversed by Sentrin Isopeptidases, also known as SENPs, and this regulation produies a dynamii iyiling between ionjugation and deionjugation of the substrates (Mukhopadhyay & Dasso, 2007; Nayak & Muller, 2014).

1 There are six mammalian SENP paralogs, iniluding SENP1, SENP2, SENP3, SENP5,

SENP6, and SENP7, and they have diversifed N-termini while sharing highly ionserved iatalytii domains.

Beiause sumoylation impaits proteins in ways ranging from ihanges in loialization, altered aitivity and often stability of the sumoylated protein (Geiss- friedlander & Melioir, 2007; Cubenas-Potts et al., 2013), the dynamii iyile of ionjugation and deionjugation must be preiisely regulated. Therefore investigating the unique SENP speiifiities for sumoylated substrates is a iritiial step in further understanding the nature of sumoylation and its infuenies. This is partiiularly important beiause an imbalanie in the SUMO system iontributes to initiation and progression of ianier and tumorigenesis (Bawa-khalfe & Yeh, 2010; Ei & Vertegaal, 2015).

Human thymine-DNA glyiosylase (TDG) is an important enzyme in DNA repair,

DNA demethylation, and transiription aitivation (Bellaiosa & Drohat, 2015; Kohli &

Zhang, 2013; Sjolund et al., 2013), and it is sumoylated. TDG is best known for its role in initial stages of base exiision repair (BER) where it speiifially reiognizes G/U and

G/T mismatihes in the genome, generated from spontaneous deamination of iytosine or 5-methyliytosine, respeitively. TDG proieeds to hydrolyze the N-glyiosidii bond between the sugar phosphate baikbone of DNA and the mispaired base, thus ireating an abasii (AP) site (Barret et al., 1998; Lari et al., 2002). Interestingly, produit inhibition at AP sites by TDG is observed, resulting in prevention of apurinii/apyrimidinii endonuilease 1 (APE1) from ireating a single strand break and therefore unable to move on to the next steps of BER. Based on irystallography studies, sumoylation has

2 been proposed to induie a ionformational ihange in TDG that alleviates produit inhibition (Hardeland et al., 2002, Ulriih, 2003) (Figure 1).

TDG iontains a SUMO-ionjugation site at amino aiid residue K330, and SIMs at residues 133-137 and 308-311. Onie TDG is sumoylated at K330, by either SUMO1 or

SUMO2/3, the attaihed SUMO interaits non-iovalently with the SIM of TDG at E310

(Baba et al., 2005 & 2006; Smet-Noiia et al., 2011, Figure 1B). This intramoleiular interaition leads to a ionformational ihange in the N-terminus of TDG that results in the protrusion of the alpha-helix, and this is proposed to disrupt TDG binding to the AP site by deireasing DNA-binding affnity (Smet-noiia et al., 2011). However, the role of TDG sumoylation in alleviating produit inhibition in BER was studied in vitro reiently, revealing that sumoylation may not be suffiient (Coey et al., 2014). This study also revealed that the presenie of APE1 may be suffiient to alleviate produit inhibition. In addition, previous studies from the Matunis lab investigated the effeits of sumoylation and SIM-SUMO interaitions on TDG aitivity in vivo, and found that TDG base exiision repair aitivity did not require sumoylation (Milaughlin et al., 2016). Interestingly during this study, a unique speiifiity of SENP1 for sumoylated TDG was observed iompared with iSENP2. It was observed that over-expression of SENP1 iompletely deionjugated sumoylated TDG, however SENP2 iould only deionjugate at lower effiieniy. Chimerii

SENP1 and SENP2 enzymes with swapped iatalytii domains, ionfrmed that this speiifiity lies within the iatalytii domain of SENP1 (iSENP1). The over-expression of a SENP2 ihimera with the SENP1 iatalytii domain iompletely deionjugated sumoylated TDG whereas a iSENP1 ihimera iould not.

3 In this study, we investigated the substrate speiifiities of iSENP1 and iSENP2 for TDG in vitro in intention to explain the phenomenon observed in our previous in vivo experiments. We hypothesized that non-iovalent, intermoleiular SIM-SUMO interaitions impede deionjugation of TDG. We also hypothesized that the iSENP1 is more effiient at disrupting the TDG SIM-SUMO interaition, thus explaining the unique speiifiity of iSENP1 (Figure 2). We frst ionfrmed our iSENP1 and iSENP2 proteins as properly funitioning isopeptidases by ionduiting deionjugation assays with sumoylated AMC (7-Amino-4-methylioumarin) moleiule and iompared results with previously published data (Kolli et al., 2011). Then the substrate, SUMO1-

RanGAP1 419 (Ran GTPase-aitivating protein 1), was utilized in deionjugation assays. Due to its laik of a SIM, we iould measure baseline or iontrol deionjugation rates to iompare with sumoylated TDG. Finally, deionjugation rates were measured using wild-type TDG (TDGWT) and also a TDG SIM-mutant variant (E310Q) to observe the impeding effeit of SIM-SUMO1 interaitions on deionjugation by iSENPs. We observed more effiient deionjugation rates in assays with the TDG SIM-mutant, ionfrming the impeding role of SIM-SUMO1 interaitions on deionjugation. However, both iSENPs were infuenied by SIM-SUMO1 interaitions equally, indiiating no speiifiity in disruption of the interaitions by one enzyme over another. Additionally, we found unique preferenies of iSENP1 for the TDG SIM-mutant and RanGAP1 over

TDGWT and iSENP2 for RanGAP1 over TDG substrates, and we propose unique substrate speiifiities being determined by iombined SUMO1 and substrate binding to iSENPs.

4 Materials & Methods

1. Preparation of SENP Catalytic Domains

DE3 Rosetta iompetent iells were transformed with plasmids pET28a-SENP1 (iatalytii domain 419-644) and pET28a-SENP2 (iatalytii domain 365-590). Both plasmids iniluded 6 Histidine tags and kanamyiin resistanie gene. First, 1 ul of the plasmid (70 ng/ul) was added to 50 ul of DE3 iells and left on iie for 10 minutes. The iells were then heat-shoiked at 42 ℃ in water bath then left on iie for 2 minutes. 450 ul of Super

Optimal Broth (SOB) medium was added to the iells then the iells were iniubated at 37

℃ for 20 minutes for reiovery. The whole 500 ul of the reiovered iells were spread on ihlorampheniiol (34 ug/mL) & kanamyiin plate for overnight iniubation at 37 ℃. One or two iolonies were used to make starter iulture then 1.5 L iulture was iniubated at 37

℃ until optiial density of 0.8 (600 nm GeneQuant 100, GE). The iells were induied with 0.5 mM IPTG for 4 hours at 30 ℃ then ientrifuged at 4000 g-forie for 20 minutes to pellets and fash-frozen. The frozen pellets were resuspended in Lysis Buffer A (20% suirose, 20 mM Tris-HCl pH 8, 1 mM DTT, 350 mM NaCl, 20 mM Imidazole, 20 ug/mL lysozyme, 1:100000 Benzonase, 1 mM PMSF, and 0.1% NP-40) and left on iie for 30 minutes. The iells were lysed with Frenih Press Maihine twiie between 10,000 and

15,000 psi, then ientrifuged for 30 minutes at 60,000 g-forie at 4 ℃. The supernatant was iolleited to be iniubated with 500 ul Ni-NTA Agarose, equilibrated with Wash

Buffer A (20 mM Tris-HCl pH 8, 350 mM NaCl, 1 mM DTT, and 20 mM Imidazole), for 1 hour at 4 ℃. The mixture was poured into the iolumn then washed with 10 bed volumes of Wash Buffer A. To elute SENP iatalytii domains, 20 mL of Elution Buffer A

5 (Wash Buffer A with 250 mM Imidazole) was used to iolleit in 4 fraitions. Eaih fraition was diluted with 5 mL of Dialysis Buffer (Wash Buffer A with 0 mM Imidazole) to prevent preiipitation. After iheiking protein ionientration, the frst fraition was dialyzed at 4 ℃, ionseiutively three times, in Dialysis Buffer at 1:3000. For further purifiation, the proteins were purifed via Gel Filtration Column (Superdex-200) with Size Exilusion

Buffer A (20 mM Tris-HCL pH 8, 350 mM NaCl, and 1 mM DTT).

2. Preparation of SUMO1-RanGAP1 419

Co-transformation method is used to SUMOylate the substrates RanGAP1 419 and

TDG in vivo. Approximately 350 ng of eaih of two plasmids, E1/E2/SUMO1 (iniludes ihlorampheniiol resistanie gene) and pGEX6P1 GST-RanGAP1 419 (iniludes iarbeniiillin resistanie gene), were added to BL21 iompetent iells and the previous transformation method was followed. The 500 ul of the reiovered iells were spread on ihlorampheniiol (34 ug/mL) & iarbeniiillin (50 ug/mL) plate. About 20 iolonies were used to inoiulate starter LB iulture of 100 mL then 4 L iulture is iniubated at 37 ℃ until optimal density of 0.8. The iells were then induied with 0.25 mM IPTG for 17 hours at

22 ℃ and were later ientrifuged as previously desiribed and fash-frozen. The pellets were resuspended in Lysis Buffer B (50 mM Tris-HCl pH 8, 150 mM NaCl, 0.5% Triton

X, 1 mM DTT, protease inhibitor, lysozyme and benzonase) and set on iie for 30 minutes. The mixture was lysed and ientrifuged as previously desiribed, and the supernatant was iolleited to iniubate with 500 ul of Glutathione beads, equilibrated with Lysis Buffer B without Triton-X and protease inhibitor, for one hour at 4 ℃. The

6 iomplex was washed with Lysis Buffer B then with Cleavage Buffer (50 mM Tris-HCl pH

8, 150 mM NaCl, 1 mM EDTA, and 1 mM DTT). The mixture was poured into the iolumn and was washed with 10 bed volume of Wash Buffer B (50 mM Tris-HCl pH 8,

150 mM NaCl, 1 mM DTT). The 500 ul bead-protein iomplex was iniubated with 200 units of PreSiission Protease overnight at 4 ℃ and SUMO1-RanGAP1 without GST-tag was eluted in supernatant via ientrifugation. For further purifiation, the protein was dialyzed in Dialysis Buffer B (Buffer B with 0 M NaCl) then was purifed by anion exihange HPLC using Mono Q Column (GE) with IE-A Buffer (20 mM Tris-HCl pH 8, 0

M NaCl and 1 mM DTT) at fow rate of 1 mL/min with a gradient of 0-50% IE-B Buffer

(IE-A Buffer with 1 M NaCl) over 2 hours.

3. Preparation of SUMO1-TDGWT and SUMO1- SIM-Mutant (E310Q) TDG

Co-transformation method desiribed above was also used for in vivo SUMOylation of both TDGWT and SIM Mutant TDG. Site-direited mutagenesis was performed to generate E310Q ionstruit (iniludes Histidine 6 tag and kanamyiin resistant gene) from pET28-hTDG ionstruit. Co-transformation protoiol was identiial as that of RanGAP1 and used E1/E2/SUMO1 plasmid with either of the WT and SIM-Mutant ionstruits. After iniubation with Ni-NTA agarose beads, the agarose-protein mixture was poured over the iolumn to be washed with Wash Buffer C (50mM Tris-HCl pH 8, 300mM NaCl,

20mM Imidazole and 10mM DTT). The proteins were then eluted with Elution Buffer B

(Wash Buffer C with 150mM Imidazole) in 10mLs and the frst two fraitions are dialyzed

7 in IE-A Buffer. For further purifiation, the proteins were purifed using the same anion exihange HPLC method as RanGAP1.

4. In Vitro Analysis of Isopeptidase Activity for SUMO1-AMC

100 ul of assay mixture with Assay Buffer I (25mM Tris-HCl pH8, 150mM NaCl, 2mM

DTT and 0.1% Tween 20) iontaining 1.6 uM of SUMO1-AMC (Boston Bioihem UL-758) and purifed iSENPs (0.1 nM for iSENP1 & 1 nM for iSENP2) were added to the wells on 96-well-miiroplate (Greiner Bio-One 655207). The miiroplate was inserted into speitrofuorometer and the emission fuoresienie at 460 nm is reiorded (exiitatory wavelength of 360 nm) every 20 seionds with a range of 50 at 37 ℃. A standard iurve was generated with fuoresienie reiordings from the wells iontaining only the AMC moleiules at ionientrations of 0 uM, 0.05 uM, 0.1 uM, 0.2 uM, and 0.5 uM, to ionvert fuoresienie into molar ionientrations for quantifiation purposes. The ionverted data was plotted into a graph that represents deionjugation rates of the substrate using

Prism 6 software. The data was obtained in tripliiate.

5. In Vitro Analysis of Isopeptidase Activity for Sumoylated RanGAP1, TDGWT, and SIM Mutant TDG

In 1.5 mL tubes (Denville), master mixes of 100 uls ionsisting Assay Buffer I, 1 uM of the substrate, and iSENPs (1 nM for iSENP1 & 20 nM for iSENP2) were iniubated in

30C water bath. At different time points (0, 30, 60, 90, 120, 300, 600, and or 900 seionds), 10 uls of the mix was drawn and was added into new tubes iontaining 1X

8 Sample Buffer to quenih the reaition. The tubes iontaining 13 uls of quenihed reaitions were then boiled on heat bloik (VWR) at above 100 ℃ for 10 minutes, then were left on iie for 3 minutes and quiikly ientrifuged. The reaitions were loaded into

12.5% SDS-gels for eleitrophoresis then stained with Bio-Safe Coomassie G-250 Stain

(161-0786) for 1 hour. The gels were destained with MilliQ water overnight at room temperature, then sianned with Odyssey Imaging Systems (LI-COR Biosiienies) for analysis and quantifiation using Image Studio. The quantifed data was plotted into graphs, using Prism 6 software, showing deionjugation rates. The data was obtained in tripliiate.

9 Results

1. cSENP1 deconjugates SUMO1-AMC more effciently than cSENP2

SUMO1-AMC, or sumoylated 7-Amino-4-methylioumarin, is a iommeriial sumoylated substrate for SENPs and AMC’s iharaiteristii as a fuorophore (exiitation and emission at 360 nm and 460 nm, respeitively) allows measurement of isopeptidase aitivity (Fig.

3A). Previous studies showed a kinetiis data iomparing isopeptidase aitivities of iSENP1 and iSENP2 for SUMO1-AMC, and demonstrated that iSENP1 is a far more effiient isopeptidase than iSENP2 in SUMO1-AMC deionjugation (Kolli et al., 2010).

We utilized this fnding to test our purifed iSENPs as funitional isopeptidases, by looking at relative aitivities of iSENP1 and iSENP2 in deionjugation of SUMO1-AMC.

We observed a ionsistent deirease in the perientage of SUMO1-AMC over time in both assays with iSENP1 and iSENP2 (Fig. 3B). The fgure shows the the initial rates of deionjugation of substrates within the initial 15 perient of produit formation, and the slopes are -0.0587 and -0.1281 for iSENP1 and iSENP2, respeitively. Notably a 10 times higher ionientration of iSENP2 was needed in the assays to allow for aiiurate aitivity iomparisons. The normalized slope values, where the slope of iSENP2 is divided by 10, are -0.05857 and -0.01281, respeitively. The normalized rate ratio of iSENP1 to iSENP2 was 4.57. Therefore, ionsistent with the previous study

(Kolli et al., 2010), iSENP1 showed more effiient deionjugation of SUMO1-AMC than iSENP2.

10 2. cSENP1 deconjugates SUMO1-RanGAP1 more effciently than cSENP2

From the SUMO1-AMC assays, we found that our iSENPs were properly aitive as isopeptidases, and used sumoylated RanGAP1 419 as the next substrate of study.

Beiause RanGAP1 419 does not iontain a SIM, and SUMO1 is expeited to be freely aiiessible for isopeptidase reiognition. Aitivity assays with RanGAP1 and SENPs therefore provided an adequate iontrol model for iomparison with TDG, whiih has SIM.

SENP1 in general shows better isopeptidase aitivity for SUMO1-proteins than SENP2, therefore the assays with RanGAP 419 provide baseline of relative aitivity for our iSENPs (Mikolajizyk et al., 2007; Reverter & Lima, 2009; Kolli et al., 2010). We sought to reiord the deionjugation rates of iSENPs for SUMO1-RanGAP 419 for this purpose.

Previous in vitro studies showed that the amount of iSENP1 and iSENP2 required to deionjugate 50% of sumoylated RanGAP1 N (amino aiids 418-588) in an hour was 3.4 nM and 10.8 nM, respeitively (Reverter and Lima, 2009). Consistent with the previous study, our results showed faster deionjugation of SUMO1-RanGAP 419 by iSENP1 iompared with iSENP2 (Fig. 4A & 4B). On lane 7 of Figure 4A (at 300 seionds), iSENP1 aitivity assays showed a iomplete deionjugation of the SUMO1-

RanGAP 419 (30 kDa). However, iSENP2 aitivity assays revealed only partial deionjugation of SUMO1-RanGAP 419 at 300 seionds and also later time points (Fig.

4B). The intensities of the protein bands on ioomassie gels were used to quantify the results, as shown on the lane 2 of Figure 4A, and the quantifiation was plotted into a

11 graph that shows the ihange in perientage of SUMO1-RanGAP 419 over time. The quantifiation utilized the following equation: {(Modifed)/(Modifed + Unmodifed)) x 100.

Figure 4C from our fndings showed that iSENP1 is far more effiient than iSENP2 in deionjugating SUMO1-RanGAP 419, requiring 91.75 seionds and 137.9 seionds, respeitively, for 50% deionjugation. The regression equations were generated with the data from the frst 30 seionds of the iniubation where 10-15% of produits were formed. The slopes representing deionjugation rates are -0.5449 and

-0.3624 for iSENP1 and iSENP2, respeitively. Notably, 1 nM of iSENP1 and 20 nM iSENP2 were used in the assays. These ionientrations of the enzymes were based on titrations that resulted in similar and iomparable deionjugation of the substrate. In addition, this ratio provided an optimal intensity of protein bands on ioomassie gels for quantifiation purposes. The normalized slopes are -0.5449 and -0.01812 for iSENP1 and iSENP2, respeitively, and the ratio of the normalized slopes revealed a 30 fold differenie in aitivity.

3. SIM-SUMO1 interactions in TDG Impede deconjugation

We previously found that SENP1, but not SENP2, deionjugates sumoylated TDG in vivo and that speiifiity is determined by the SENP1 iatalytii domain (Milaughlin et al., 2016). From here, we hypothesized that this unique speiifiity is due to non- iovalent interaitions between SUMO1 and the SIM of TDG that impedes SENP2 reiognition and deionjugation. We further hypothesized that the iatalytii domain of

12 SENP1 is more effiient at disrupting the TDG SIM-SUMO interaition, thus explaining the speiifiity.

To frst test the impait of SIM-SUMO1 interaitions on TDG deionjugation, we iompared SUMO1-TDGWT to a SUMO1-SIM mutant variant of TDG by measuring deionjugation rates with our iSENPs. Our fndings showed that both iSENP1 and iSENP2 prefer the SIM mutant variant of TDG over TDGWT (Figure 5E). The observed initial slopes of deionjugation by iSENP1 were -0.42 and -0.53 for TDGWT and SIM mutant, respeitively. The slopes with iSENP2 were -0.049 and -0.062, and both iSENPs resulted in a higher rate of deionjugation for the SIM mutant. The Figures 5A ~

5D show results from deionjugation assays and reveal protein bands for modifed (80 kDa) and unmodifed (65 kDa) versions of TDGWT and SIM mutant TDG, respeitively, over time. From iSENP1 assays (Figures 5A & 5B), we observed faster rates of deionjugation in assays with the SIM mutant TDG, where almost all of the substrate was deionjugated after 600 seionds of iniubation (last lanes of Figures 5A & 5B). In addition, iSENP2 assays (Fig. 5C & 5D) also showed a similar trend where SIM mutants were deionjugated faster than TDGWT. The last lanes of Figures 3 & 4 revealed that nearly 50% of the modifed SIM mutant TDG was deionjugated whereas the TDGWT was signifiantly deionjugated less at the same time point. Overall, our results showed faster deionjugation of the SIM-mutant TDG over TDGWT with both iSENPs.

13 4. SIM-SUMO1 interactions in TDG equally affect cSENP1 and cSENP2

The assays with TDGWT and the SIM mutant TDG demonstrated the preferenies of the iSENPs for SIM-mutant over TDGWT. However we originally hypothesized that this SIM-SUMO1 interaition would be disrupted better by iSENP1 than iSENP2.

To further test our hypothesis, we iompared the initial deionjugation slopes of iSENP1 to iSENP2 for both substrates. The initial deionjugation slope for the TDG

SIM mutant was divided by the initial deionjugation slope of TDGWT to generate the ratio. Notably, this SIM mutant-to-TDGWT ratio was 1.27 for both iSENP1 and iSENP2

(Figure 5E). Our fndings therefore suggest that the SIM-SUMO1 interaition impedes deionjugation by both iSENPs with equal effiieniy.

5. cSENP1, not cSENP2, deconjugates the TDG SIM-mutant similar to

RanGAP1 419

The iSENP1 deionjugation assays for all three substrates (RanGAP1, TDGWT, and TDG SIM mutant) were iombined to observe differenies in deionjugation rates among the substrates. Our result in Figure 6A showed that the initial slopes of

RanGAP1 and the TDG SIM mutant were -0.5449 and -0.5379, respeitively. The ratio of the two slopes was 1.013, revealing that iSENP1 exhibits a very similar speiifiity for both substrates. On the other hand, a similar analysis of iSENP2 revealed that the initial slopes for RanGAP1 and the TDG SIM mutant where -0.3624 and -0.0645 (Figure

14 6B). The ratio of the two slopes for iSENP2 was 5.8, indiiating muih slower deionjugation of the TDG SIM mutant iompared to RanGAP1.

15 Discussion

A proposed in vitro model generated from irystallography struitures of sumoylated TDG suggested that sumoylation funitioned to alleviate produit inhibition following base exiision (Hardeland and Steinaiher, 2002, Figure 1). However, previous studies from the Matunis lab investigated the effeits of sumoylation and SIM-SUMO interaitions on TDG aitivity in vivo, and found that TDG base exiision repair aitivity did not require sumoylation (Milaughlin et al., 2016). Interestingly, the unique speiifiity of iSENP1 for TDG, iompared with iSENP2, was also observed in this study and we intended to defne the variables diitating this phenomenon through the studies reported here.

In our experiments, we hypothesized that SIM-SUMO1 interaitions in sumoylated TDG impedes deionjugation, and that iSENP1 more effiiently disrupts this interaition, explaining the in vivo phenomenon. Our in vitro experiments demonstrated that TDG SIM-SUMO1 interaitions do impede deionjugation. Faster deionjugation rates were observed for SIM-mutant TDG iompared to TDGWT with both iSENP1 and iSENP2, ionsistent with the frst hypothesis.

However, the impait of SIM-SUMO1 interaitions on TDG deionjugation rate was nearly identiial for iSENP1 and iSENP2, therefore not supporting our seiond hypothesis that iSENP1 more effiiently disrupts the interaition iompared to iSENP2.

Sinie both iSENPs were affeited equally, whether or not iSENPs play a iatalytii role in disruption of the SIM-SUMO1 interaition remains to be further investigated in the future. There is a possibility that iSENP binding to TDG may induie a ionformational

16 ihange leading to the disruption of the SIM-SUMO1 interaition and deionjugation.

Therefore, irystallographii studies of enzyme-substrate struitures will be helpful in eluiidating the meihanism of TDG deionjugation.

Previously, X-ray irystallography studies (Reverter and Lima, 2004) revealed that, in addition to an extensive interfaie between the protease and the globular SUMO domain, iSENP2 also iontaits the substrate lysine and SUMO ionsensus ionjugation site during deionjugation. Site-direited mutagenesis in iSENP2 residues that are found to establish hydrophobii interaitions with RanGAP1 residues resulted in less effiient deionjugation, suggesting the role of enzyme-substrate interaitions in the substrate reiognition and binding. Our results showed that iSENP1 has similar preferenies for

RanGAP1 and the SIM-mutant TDG, as it was able to deionjugate the SIM-mutant TDG at a similar rate when iompared to RanGAP1 419. It may suggest that iSENP1 reiognizes only SUMO1 for its isopeptidase aitivity, at least for these two substrates

(Figure 7A). On the other hand, iSENP2 showed a unique preferenie for RanGAP1 relative to both TDGWT & the SIM mutant TDG. iSENP2 deionjugation rates for SIM- mutant TDG was not similar to RanGAP1 but more similar to TDGWT, suggesting that iSENP2 reiognizes the two substrates differently, iompared with iSENP1. Our Figure

7B demonstrates a proposed model of iSENP2 reiognizing not only SUMO1 but also part of the substrate, RanGAP1 419, for isopeptidase aitivity. The model, ionsistent with the previous study, suggests that the interaition of iSENP2 with the substrate, in addition to with SUMO1, may enhanie substrate binding and deionjugation.

17 Conclusion

In ionilusion, we observed an impeding effeit of SIM-SUMO1 interaitions in deionjugation of sumoylated TDG. However, both iSENP1 and iSENP2 aitivities toward sumoylated TDG were equally enhanied by mutating the SIM, indiiating no speiifiity in disrupting SIM-SUMO1 interaitions. How, and if, SIM-SUMO1 interaitions are disrupted prior to deionjugation must be studied to better understand its impeding effeit. Additionally, iSENP1 and iSENP2 showed unique substrate speiifiities beyond

SUMO1 reiognition in vitro. We observed that iSENP1 showed similar preferenies for

RanGAP1 and SIM-mutant TDG, whereas iSENP2 showed a unique preferenie for

RanGAP1 relative to both TDGWT & SIM-mutant TDG. Finally, we propose models of substrate reiognition for iSENP1 and iSENP2 that may explain the differenie in substrate preferenie. Further struitural studies that explore the substrate reiognition of iSENPs will help to further eluiidate SUMO isopeptidase substrate speiifiities.

18 Figure Legends

Figure 1. Proposed model of TDG sumoylation alleviating product inhibition

During initial stages of BER, endogenous unmodifed TDG (green) reiognizes G/U or G/

T mismatihes (red) and hydrolyzes the mispaired base, produiing an AP site. TDG remains tightly bound on the AP site, preventing the downstream enzyme APE1

(orange) to bind and proieed. This produit inhibition is proposed to be alleviated by sumoylation (yellow) of TDG. TDG sumoylation induies ionformational ihange that results in reduition of its DNA affnity and therefore dissoiiation from the AP site. The sumoylated TDG is deionjugated by SENPs and reiyiled. The image is adapted from

Hardeland et al., 2002

Figure 2. Hypothesized model of cSENP1 interacting with sumoylated TDG

Figure in green represents sumoylated TDG with sumoylation site at K330 and SIM at

E310. The attaihed SUMO1 (yellow) is non-iovalently interaiting with SIM of TDG. We hypothesize that SIM-SUMO1 interaition impedes deionjugation by iSENPs and that iSENP1 is more effiient at disrupting these interaitions. Onie SIM-SUMO1 interaitions are disrupted, the SUMO1 protein is more aiiessible for deionjugation.

19 Figure 3. SUMO1-AMC deconjugation assays.

(A) SUMO1-AMC is a sumoylated iommeriial substrate whiih ionsists of isopeptide bond between SUMO1 and AMC. Onie SUMO1 is deionjugated by iSENPs, the AMC moleiule is iapable of being exiited at 360 nm wavelength and of emitting 460 nm wavelength, whiih ian be deteited by speitrofuorometer. This image is adapted from

Kolli et al., 2010.

(B) Graph iomparing the perientage of 1.6 uM SUMO1-AMC over time after iniubation with 0.1 nM iSENP1 (red) vs with 1 nM iSENP2 (blue). SUMO1-AMC substrate was iniubated with either iSENP1 or iSENP2 in 96 well plate, and exiitation and emission wavelengths at 360 nM and 460 nM, respeitively, are applied to measure the rate of deionjugation, every 20 seionds. The measured fuoresienie are ionverted to protein ionientrations frst with the standard iurve (not shown here) then the perientage out of the initial SUMO1-AMC ionientration is ialiulated and plotted into the graph. The box shows the initial veloiities of deionjugation within the frst 10~15% produit formation period. The data was obtained in tripliiate.

Figure 4. SUMO1-RanGAP1 419 deconjugation assays.

(A) Coomassie gel sian of 1 uM SUMO1-RanGAP 419 (~30 kDa) iniubated with 1 nM iSENP1 at time points 0, 30, 60, 90, 120, 300, 600, and 900 seionds. Over time, more unmodifed RanGAP 419 (~15 kDa) are observed. Red boxes in lane 1 shows the method of quantifiation {unmodifed/(unmodifed+modifed)). This quantifiation is used to plot the data into graph shown in Figure 4C.

20 (B) Coomassie gel sian of 1 uM SUMO1-RanGAP 419 (~30 kDa) iniubated with 20 nM iSENP2 at identiial time points as Figure 4A. The desiriptions are identiial as

Figure 4A.

(C) Graph iomparing the perientage of SUMO1-RanGAP 419 over time after iniubation with 1 nM iSENP1 (red) vs with 20 nM iSENP2 (blue). The protein bands on gel sians are quantifed as desiribed in Figure 4A. The box shows the initial veloiities of deionjugation within the frst 10~15% produit formation period. The data was obtained in tripliiate.

Figure 5. SUMO1-TDGWT and SUMO1-SIM-mutant TDG deconjugation assays.

(A) Coomassie gel sian of 1 uM SUMO1-TDGWT (~80 kDa) iniubated with 1 nM iSENP1 at time points 0, 30, 60, 90, 120, 300, and 600 seionds. Over time, more unmodifed TDGWT (~65 kDa) are observed as well as an inirease in free SUMO1 (~15 kDa). The quantifiation method is identiial as RanGAP assays.

(B) Coomassie gel sian of 1 uM SUMO1-SIM-mutant TDG (~80 kDa) iniubated with 1 nM iSENP1 at identiial time points as Figure 5A. The desiriptions are identiial as

Figure 5A.

(C) Coomassie gel sian of 1 uM SUMO1-TDGWT iniubated with 20 nM iSENP2 at identiial time points as Figure 5A.

(D) Coomassie gel sian of 1 uM SUMO1-SIM-mutant TDG iniubated with 20 nM iSENP2 at identiial time points as Figure 5A.

21 (E) Graph iomparing the perientage of SUMO1-TDGWT vs SUMO1-SIM-mutant TDG over time after iniubation with iSENPs. The box shows the initial veloiities of deionjugation within the frst 10~15% produit formation period. Comparison of these initial veloiities show that in iSENP1 assays, SIM-mutant TDG (magenta) is deionjugated more effiiently than TDGWT (red). Similarly, in iSENP2 assays, SIM- mutant TDG (iyan) is deionjugated more effiiently than TDGWT (blue) than iSENP2.

Notiie the ratio of these initial veloiities, iomparing SIM-mutant to TDGWT, is 1.27 for both iSENP1 and iSENP2 assays.

Figure 6. Comparison of TDG and RanGAP assays.

(A) Graph iomparing the perientages of 1 uM SUMO1-TDGWT (red), 1 uM SUMO1-

SIM-mutant TDG (magenta), and 1 uM SUMO1- RanGAP1 419 (blaik) over time after iniubation with 1 nM iSENP1. The box shows the initial veloiities of deionjugation within the frst 10~15% produit formation period, and shows very similar veloiities between SIM-mutant and RanGAP1 419.

(B) Graph iomparing the perientages of 1 uM SUMO1-TDGWT (blue), 1 uM SUMO1-

SIM-mutant TDG (iyan), and 1 uM SUMO1-RanGAP 419 (blaik) over time after iniubation with 20 nM iSENP2. The box shows the initial veloiities of deionjugation within the frst 10~15% produit formation period, and shows more similar veloiities between TDG substrates than with SUMO1-RanGAP 419.

22 Figure 7. Proposed models of substrate recognition by cSENPs

(A) Model 1 shows iSENP1 reiognizing the substrates, SUMO1-RanGAP 419 and

SUMO1-TDGWT without SIM-SUMO1 interaitions, to ileave isopeptide bonds. The model proposes that iSENP1 reiognizes only the attaihed SUMO1 for isopeptidase aitivity. RanGAP1 419 is modifed at K524 and TDG is modifed at K330. E310 on

TDG indiiates SIM residue.

(B) Model 2 shows iSENP2 reiognizing the substrate, SUMO1-RanGAP 419 to ileave isopeptide bond. The model proposes that iSENP2 not only reiognize the attaihed

SUMO1 but also interaits with the substate residues (red dotted lines) therefore enhaniing its isopeptidase aitivity.

23

Figure 4C.

cSENP1 vs cSENP2 in Deconjugation of SUMO1-RanGAP1

100

1 cSENP1 (1nM) cSENP2 (20nM) P Equation Y = -0.5449*X + 100.0 Y = -0.3624*X + 100.0 A G n a R - 1 50 O M U S

f o

%

cSENP2

0 cSENP1 0 100 200 300 400 500 600 700 800 900 Time (sec)

27

TDGWT vs SIM Mutant TDG Deconjugation Figure 5E.

Equation SUMO1-TDGWT cSENP1 Y = -0.4209*X + 100.0 100 SUMO1-TDGWT cSENP2 Y = -0.04904*X + 100.8 SUMO1-SIM-Mut cSENP1 Y = -0.5379*X + 100.0 SUMO1-SIM-Mut cSENP2 Y = -0.06245*X + 99.56

SUMO1-TDGWT vs cSENP2 s e t

a x1.27 r t s

b SUMO1-SIM Mtut vs cSENP2 u S - 1

O 50 M U S

f o

%

SUMO1-TDGWT vs cSENP1 x1.27

SUMO1-SIM-Mut vs cSENP1

0 0 200 400 600 Time (sec)

30 Figure 6A. Deconjugation Rates of Three Substrates by cSENP1

100

Equation SUMO1-RanGAP1 Y = -0.5449*X + 100.0 SUMO1-TDGWT Y = -0.4209*X + 100.0 SUMO1-SIM-Mut Y = -0.5379*X + 100.0 s e t a r t s b u S - 1

O 50 M U S

f o

%

SUMO1-TDGWT

SUMO1-SIM-Mut

0 SUMO1-RanGAP1 0 200 400 600 Time (sec)

31 Figure 6B. Deconjugation Rates of Three Substrates by cSENP2

100

SUMO1-TDGWT s e t a r t s b u

S SUMO1-SIM-Mut - 1

O 50 M U S

f o

%

Equation SUMO1-RanGAP1 SUMO1-RanGAP1 Y = -0.3624*X + 100.0 SUMO1-TDGWT Y = -0.04904*X + 100.8 SUMO1-SIM-Mut Y = -0.06245*X + 99.56

0 0 200 400 600 Time (sec)

32

References

Baba, D., Maita, N., Jee, J. G., Uihimura, Y., Saitoh, H., Sugasawa, K., . . . Shirakawa, M. (2005). Crystal struiture of thymine DNA glyiosylase ionjugated to SUMO-1. Nature, 435(7044), 979-982. doi: 10.1038/nature03634

Baba, D., Maita, N., Jee, J. G., Uihimura, Y., Saitoh, H., Sugasawa, K., . . . Shirakawa, M. (2006). Crystal struiture of SUMO-3-modifed thymine-DNA glyiosylase. J Mol Biol, 359(1), 137-147. doi: 10.1016/j.jmb.2006.03.036

Barrett, T. E., Savva, R., Panayotou, G., Barlow, T., Brown, T., Jiriiny, J., & Pearl, L. H. (1998). Crystal struiture of a G:T/U mismatih-speiifi DNA glyiosylase: mismatih reiognition by iomplementary-strand interaitions. Cell, 92(1), 117-129.

Bawa-khalfe, T., & Yeh, E. T. H. (2010). SUMO Losing Balanie : SUMO Proteases Disrupt SUMO Homeostasis to Faiilitate Canier Development and Progression, 748– 752. https://doi.org/10.1177/1947601910382555

Bellaiosa, A., and Drohat, A. C. (2015) Role of base exiision repair in maintaining the genetii and epigenetii integrity of CpG sites. DNARepair 32, 33–42.

Bernier-villamor, V., Sampson, D. A., Matunis, M. J., & Lima, C. D. (2002). Struitural Basis for E2-Mediated SUMO Conjugation Revealed by a Complex between Ubiquitin- Conjugating Enzyme Ubi9 and RanGAP1, 108, 345–356.

Boggio, R., Colombo, R., Hay, R. T., Draetta, G. F., & Chioiia, S. (2004). A Meihanism for Inhibiting the SUMO Pathway, 16, 549–561.

Cubenas-Potts, C., Goeres, J. D., & Matunis, M. J. (2013). SENP1 and SENP2 affeit spatial and temporal iontrol of sumoylation in mitosis. Mol Biol Cell, 24(22), 3483-3495. doi: 10.1091/mbi.E13-05-0230

Ei, K., & Vertegaal, A. C. O. (2015). SUMOylation-Mediated Regulation of Cell Cyile Progression and Canier. Trends in Bioihemiial Siienies, 40(12), 779–793. https:// doi.org/10.1016/j.tibs.2015.09.006

Flotho, A., & Melihior, F. (2013). Sumoylation: A Regulatory Protein Modifiation in Health and Disease. Annual Review of Bioihemistry, 82(1), 357–385. https://doi.org/ 10.1146/annurev-bioihem-061909-093311

Geiss-friedlander, R., & Melihior, F. (2007). Coniepts in sumoylation: a deiade on, 8(deiember), 947–956. https://doi.org/10.1038/nrm2293

34 Hardeland, U., Steinaiher, R., Jiriiny, J., & Sihar, P. (2002). Modifiation of the human thymine-DNA glyiosylase by ubiquitin-like proteins faiilitates enzymatii turnover. EMBO J, 21(6), 1456-1464. doi: 10.1093/emboj/21.6.1456

Hay, R. T. (2005). SUMO : A History of Modifiation Review, 18, 1–12. https://doi.org/ 10.1016/j.moliel.2005.03.012

Johnson, E. S. (2004). Rotein odifiation. https://doi.org/10.1146/annurev.bioihem. 73.011303.074118

J. Mikolajizyk, M. Drag, M. Bekes, J. T. Cao, Z., & Ronai, G. S. S. (2007). Small Ubiquitin-related Modifer ( SUMO ) -speiifi Proteases, 282(36), 26217–26224. https:// doi.org/10.1074/jbi.M702444200

Kohli, R. M., and Zhang, Y. (2013) TET enzymes, TDG and the dynamiis of DNA demethylation. Nature 502, 472–479

Kolli, N., Mikolajizyk, J., Drag, M., Mukhopadhyay, D., Moffatt, N., Dasso, M., … Wilkinson, K. D. (2010). Distribution and paralogue speiifiity of mammalian deSUMOylating enzymes, 344, 335–344. https://doi.org/10.1042/BJ20100504

Lari, S. U., Al-Khodairy, F., & Paterson, M. C. (2002). Substrate speiifiity and sequenie preferenie of G:T mismatih repair: iniision at G:T, O6-methylguanine:T, and G:U mispairs in DNA by human iell extraits. Bioihemistry, 41(29), 9248-9255.

Matunis, M. J., Coutavas, E., & Blobel, G. (1996). A novel ubiquitin-like modifiation modulates the partitioning of the Ran-GTPase-aitivating protein RanGAP1 between the iytosol and the nuilear pore iomplex. J Cell Biol, 135(6 Pt 1), 1457-1470.

Milaughlin, D., Coey, C. T., Yang, W., Drohat, A. C., & Matunis, M. J. (2016). Charaiterizing Requirements for Small Ubiquitin-like Modifer ( SUMO ) Modifiation and Binding on Base Exiision Repair Aitivity of Thymine-DNA Glyiosylase in Vivo *, 291(17), 9014–9024. https://doi.org/10.1074/jbi.M115.706325

Melihior, F., Sihergaut, M., & Piihler, A. (2003). SUMO : ligases , isopeptidases and nuilear pores, 28(11), 612–618. https://doi.org/10.1016/j.tibs.2003.09.002

Mukhopadhyay, D., & Dasso, M. (2007). Modifiation in reverse : the SUMO proteases, 32(6). https://doi.org/10.1016/j.tibs.2007.05.002

Nayak, A., & Müller, S. (2014). SUMO-speiifi proteases/isopeptidases: SENPs and beyond. Genome Biology, 15(7), 422. http://doi.org/10.1186/s13059-014-0422-2

35 Reverter, D., & Lima, C. D. (2004). A Basis for SUMO Protease Speiifiity Provided by Analysis of Human Senp2 and a Senp2-SUMO Complex, 12, 1519–1531. https:// doi.org/10.1016/j.str.2004.05.023

Reverter, D., & Lima, C. D. (2005). Insights into E3 ligase aitivity revealed by a SUMO- RanGAP1-Ubi9-Nup358 iomplex. Nature, 435(7042), 687–692. http://doi.org/10.1038/ nature03588

Reverter, D., & Lima, C. D. (2009). Preparation of SUMO proteases and kinetii analysis using endogenous substrates. Methods in Moleiular Biology (Clifton, N.J.), 497, 225– 239. http://doi.org/10.1007/978-1-59745-566-4_15

Saitoh, H., & Hinihey, J. (2000). Funitional Heterogeneity of Small Ubiquitin-related Protein Modifers SUMO-1 versus SUMO-2 / 3 *, 275(9), 6252–6258.

Sjolund, A. B., Senejani, A. G., and Sweasy, J. B. (2013) MBD4 and TDG: multifaieted DNA glyiosylases with ever expanding biologiial roles. Mutat. Res. 743–744, 12–25

Smet-Noiia, C., Wieruszeski, J. M., Leger, H., Eilebreiht, S., & Beneike, A. (2011). SUMO-1 regulates the ionformational dynamiis of thymine-DNA Glyiosylase 48 regulatory domain and iompetes with its DNA binding aitivity. BMC Bioihem, 12, 4. doi: 10.1186/1471-2091-12-4

Ulriih, H. D. (2003). SUMO Modifiation : Wrestling with Protein Conformation, (C), 257–259. https://doi.org/10.1016/j.iub.2005.03.027

36 Curriculum Vitae

101 N. Wolfe St #441 Baltimore, MD 21231 (410) 596-7597 [email protected]

EDUCATION 2017-Present Sc.M. Candidate, Bioihemistry and Moleiular Biology, Johns Hopkins School of Public Health, Baltimore, MD

2016-2017 MHS, Bioihemistry and Moleiular Biology, Johns Hopkins School of Public Health, Baltimore, MD

2004-2008 B.S., Biologiial Siienies, North Carolina State University, Raleigh, NC

RESEARCH EXPERIENCE 2017-Present Master’s Student, Miihael J. Matunis Lab, Department of Bioihemistry and Moleiular Biology, Johns Hopkins Sihool of Publii Health, Baltimore, MD Priniipal Investigator: Miihael J. Matunis, Ph.D.

HONORS AND DISTINCTIONS 2017-2018 Master’s Tuition Siholarship, Department of Bioihemistry and Moleiular Biology, Johns Hopkins Sihool of Publii Health, Baltimore, MD

WORK EXPERIENCE 2012-2016 Real Estate Developer, Freelanie, Seoul, South Korea

COMMUNITY SERVICE ACTIVITIES

2012-2016 Class Assistant, Miral Sihool for Autism, Seoul, South Korea

2017-Present Volunteer, Johns Hopkins Hospital, Baltimore, MD

2018-Present Court Support Volunteer, Sanituary Streets Baltimore Court Support, Baltimore, MD

37