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Plasmid Replication-Associated Single-Strand-Specific 12858–12873 Nucleic Acids Research, 2020, Vol. 48, No. 22 Published online 3 December 2020 doi: 10.1093/nar/gkaa1163 Plasmid replication-associated single-strand-specific methyltransferases Alexey Fomenkov 1,*, Zhiyi Sun1, Iain A. Murray 1, Cristian Ruse1, Colleen McClung1, Yoshiharu Yamaichi 2, Elisabeth A. Raleigh 1,* and Richard J. Roberts1,* 1New England Biolabs Inc., 240 County Road, Ipswich, MA, USA and 2Universite´ Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France Downloaded from https://academic.oup.com/nar/article/48/22/12858/6018438 by guest on 24 September 2021 Received August 06, 2020; Revised November 10, 2020; Editorial Decision November 11, 2020; Accepted November 12, 2020 ABSTRACT RM-associated modification and the diversity of associ- ated functions remains incompletely understood (3). Long- Analysis of genomic DNA from pathogenic strains read, modification-sensitive SMRT sequencing technology of Burkholderia cenocepacia J2315 and Escherichia has facilitated sequencing and assembly of a wide variety coli O104:H4 revealed the presence of two unusual of genomes and also clarified the modification repertoire. MTase genes. Both are plasmid-borne ORFs, carried Detection of the modification status of bases is possible by pBCA072 for B. cenocepacia J2315 and pESBL for for m6A, m4C and oxidized forms of m5C modified bases E. coli O104:H4. Pacific Biosciences SMRT sequenc- (m5hC and 5caC) (4). Recently, high throughput analy- ing was used to investigate DNA methyltransferases sis of 230 diverse bacterial and archaeal methylomes strik- M.BceJIII and M.EcoGIX, using artificial constructs. ingly revealed that almost 50% of organisms harbor Type Mating properties of engineered pESBL derivatives II DNA methyltransferases (MTase) homologs with no ap- were also investigated. Both MTases yield promis- parent cognate restriction enzyme (RE) (5). These ‘orphan’ MTases sometimes exhibit patterns of in- cuous m6A modification of single strands, in the = = complete methylation that distinguish them from RM sys- context SAY (where S CorGandY CorT). tem MTases (6,7), enabling roles in regulation of gene ex- Strikingly, this methylation is asymmetric in vivo,de- pression and DNA replication in diverse bacterial and ar- tected almost exclusively on one DNA strand, and chaeal phyla. Two well-studied examples in Escherichia coli is incomplete: typically, around 40% of susceptible are the orphan DNA MTases Dam and Dcm, which play motifs are modified. Genetic and biochemical stud- roles in mismatch DNA repair, DNA replication and phase ies suggest that enzyme action depends on repli- variation of protein expression (6). In alpha-proteobacteria, cation mode: DNA Polymerase I (PolI)-dependent Caulobacter crescentus CcrM is also well studied, and ColE1 and p15A origins support asymmetric mod- evolved independently to regulate numerous cellular func- ification, while the PolI-independent pSC101 origin tions (e.g. (8,9)). Additional examples are accumulating: does not. An MTase-PolI complex may enable dis- M.CsaII modifies 76% of sites in the native host10 ( ), M.EcoGX modifies 34% (11). Selective modification by the crimination of PolI-dependent and independent plas- conserved CamA MTase is also widespread in the popula- mid origins. M.EcoGIX helps to establish pESBL in tion of C. difficile (12). new hosts by blocking the action of restriction en- Genome and methylome analysis of total DNA from two zymes, in an orientation-dependent fashion. Expres- pathogenic strains of Burkholderia cenocepacia J2315 and sion and action appear to occur on the entering sin- E. coli O104:H4 revealed the presence of two related and ap- gle strand in the recipient, early in conjugal transfer, parently silent MTases not previously characterized. Both until lagging-strand replication creates the double- are coded by plasmid-borne ORFs, in pBCG2315 for B. stranded form. cenocepacia J2315 (13,14) and pESBL for E. coli O104:H4 (11). Genetic and biochemical study of these two MTases led INTRODUCTION us to propose that they modify single-stranded DNA in co- The role of DNA modification in restriction-modification ordination with DNA Polymerase I in vivo.Herewepresent (RM) mechanisms of prokaryotic cells was established a short introduction to replicons used for this study and the >50 years ago (1,2). However, the prevalence of non- in vivo mechanisms that reveal ssDNA substrates. *To whom correspondence should be addressed. Tel: +1 978 380 7560; Fax: +1 978 921 1350; Email: [email protected] Correspondence may also be addressed to Elisabeth A. Raleigh. Email: [email protected] Correspondence may also be addressed to Richard J. Roberts. Email: [email protected] C The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Research, 2020, Vol. 48, No. 22 12859 Vegetative plasmid replication modes The recipient cell frequently regulates DNA entry with defense strategies, including restriction activities and SOS- Plasmid families have characteristic replication-initiation regulated suicide mechanisms. The gene neighborhood in modules (ori regions, also called replicons) (15) with typical the early-transferred conjugal DNA includes many anti- copy number per chromosome within the host. Host repli- defense factors that thwart these cellular regulators of DNA cation machinery is used for elongation, so the ori regions exchange: ArdA, which inhibits action of Type I RM sys- can be thought of as adaptors to a common downstream tems; PsiA and PsiB, which suppress induction of the output. SOS response (and resident prophages) by entering single Plasmids with ColE1 and p15A replication origins (used strands; ParB homologs, and Ssb proteins, which bind to here) use a PolI-dependent replication initiation mechanism single strands (27). These functions are always coded for in common to many high-copy plasmids of Proteobacteria the same orientation relative to oriT ensuring they are tran- (16). The host’s vegetative RNA polymerase synthesizes an scribed and translated from the single-strand that is trans- initiator RNA (RNAII) and, from the opposite strand, a Downloaded from https://academic.oup.com/nar/article/48/22/12858/6018438 by guest on 24 September 2021 ferred into the recipient (28). Expression requires the recip- copy-number regulator (RNAI). The RNAII:DNA hybrid ient RNAP (29). M.EcoGIX and its homologs are found stably displaces the parental leading strand. The hybrid is here, in the orientation that allows expression from the anti- processed by RNAse H to provide the primer for DNA ex- defense promoters. tension by PolI. If RNAI anneals to RNAII, processing Complete establishment for conjugal plasmids requires and primer extension are prevented, lowering the number resumption of ds→ds replication. Willetts and Wilkins (30) of plasmid copies. RNAII initiation is regulated by Dam point out that at least two recipient priming mechanisms modification of the promoter (17). This replication is resis- are needed for F’ transfer and establishment, one RifR in tant to translation-inhibiting drugs, making it effectively in- a wild-type recipient (aka replisome assembly with PolIII) dependent of cellular mechanisms that coordinate division and one RifS in a DnaB-defective host (Clamp helicase, so and replication (18). Plasmids with distinct sequences in the replisome-deficient). Complete transfer required DnaE (Pol RNAI-RNAII annealing region do not cross-inhibit, and III). thus are compatible, even though both rely on the same ini- tiation mechanism. ColE1 and p15A are compatible for this reason. Phage M13 biology Though replicon classification is incomplete (19), both Single-strand DNA phages develop through ssDNA and ds- low-copy pSC101 (used here) and many characterized con- DNA life phases (31). M13 filamentous virions comprise a jugal plasmids (including the F factor) employ plasmid- circular single-stranded DNA molecule encased in a thin encoded initiator proteins. These proteins promote assem- flexible tube made up of protein VIII, with protein IIIat bly of the DNA Polymerase III replisome during vegetative one end. The F factor pilus is required by pIII as a recep- replication (15). Like the host cell, pSC101 requires only the tor. Once inside the cell, replication occurs in three phases; → 5 3 Exonuclease activity of PolI, for Okazaki fragment establishment (ss→ds), amplification (ds-ds), and secretion maturation (20) but not the polymerase activity. Prolonged (ds→ss). For viral secretion, the phage employs a rolling exposure of single-strand regions has not been reported for circle replication mechanism, with virions assembled on the such replication strategies. displaced single strand, which are then secreted through the cell envelope. Replication during viral secretion is mediated by PolIII, initiating at nicks catalyzed by gpII of M13 (31). Conjugal replication in the donor and the recipient Replication does not require the polymerase activity of PolI In addition to vegetative replication during cell growth, con- (see e.g. (32)) but does require the 5 →3 exonuclease activ- jugal plasmids use a distinct replication program to move ity (33,34). This differs in important ways from the theta- from one cell
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