10216–10229 Nucleic Acids Research, 2016, Vol. 44, No. 21 Published online 17 August 2016 doi: 10.1093/nar/gkw720 Incision of damaged DNA in the presence of an impaired Smc5/6 complex imperils genome stability Jie Peng and Wenyi Feng*
Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, NY 13210, USA
Received February 01, 2016; Revised August 05, 2016; Accepted August 08, 2016
ABSTRACT cleotide excision repair (RER) pathways, respectively (7). / Virtually all DNA repair pathways require recognition and The Smc5 6 complex is implicated in homologous subsequent excisions of the damaged nucleotide, giving rise recombination-mediated DNA repair during DNA to a single-stranded gap in the DNA polymer (7). The mech- damage or replication stress. Here, we analysed anisms ensuring such a structural alteration of the chro- genome-wide replication dynamics in a hypomorphic mosome is efficiently repaired and does not lead to further budding yeast mutant, smc6-P4. The overall replica- degradation are underexplored. Conceivably, these mecha- tion dynamics in the smc6 mutant is similar to that nisms would involve protein complexes directly interacting in the wild-type cells. However, we captured a dif- with the chromosomal DNA and implicated in DNA dam- ference in the replication profile of an early S phase age or replication stress response. sample in the mutant, prompting the hypothesis that One of the three evolutionarily conserved Structure the mutant incorporates ribonucleotides and/or ac- Maintenance of Chromosome (SMC) protein complexes, the Smc5/6 complex, plays a key role in DNA damage re- cumulates single-stranded DNA gaps during replica- pair. The Smc5 and Smc6 proteins, which form the het- tion. We tested if inhibiting the ribonucleotide exci- erodimeric core, together with six other essential Non- sion repair pathway would exacerbate the smc6 mu- SMC Element 1–6 (Nse1-6) constitute the Smc5/6 com- tant in response to DNA replication stress. Contrary plex (8–16). Cells with mutations in any subunit of the to our expectation, impairment of ribonucleotide ex- Smc5/6 complex are hypersensitive to various reagents in- cision repair, as well as virtually all other DNA re- cluding hydroxyurea (HU, a ribonucleotide reductase in- pair pathways, alleviated smc6 mutant’s hypersen- hibitor), methyl methanesulfonate (MMS, an alkylating sitivity to induced replication stress. We propose reagent) and ultraviolet light (9,12,15–28). Cruciform DNA that nucleotide incision in the absence of a func- molecules (X-molecules) representing abnormal recombi- / tional Smc5/6 complex has more disastrous out- nation intermediates are observed in MMS-treated smc5 6 comes than the damage per se. Our study provides mutants (24,29). Removal of recombinogenic proteins such as Rad51, Mph1, Mms2 and Shu1 decreases the level of X- novel perspectives for the role of the Smc5/6com- molecules in smc5/6 cells and rescues their hypersensitiv- plex during DNA replication. ity to replication stress-inducing reagents (24,29,30). Thus, it was established that the key function(s) of the Smc5/6 INTRODUCTION complex are preventing and/or resolving abnormal recom- Eukaryotic DNA replication follows a temporal program bination intermediates during replication stress. Consistent where replication origins activate in a staggered order to cre- with this notion loss of a functional Smc5/6 complex can ate a continuum of replication forks progressing through the lead to aberrant chromosome segregation during mitosis chromosome before encountering another convergent fork and meiosis (31–33). Removal of the joint molecule in- (1,2). Eukaryotic DNA polymerases responsible for proces- termediates in meiosis via the XPF-family endonuclease, sive synthesis have intrinsic nucleotide mis-incorporation Mus81-Mms4/Eme1, requires a functional Smc5/6 com- of not only mismatched deoxyribonucleoside monophos- plex (34,35). Finally, the Smc5/6 complex counteracts repli- phates (dNMPs) but also ribonucleoside monophosphates cation fork regression (a source of X-molecules) by the (rNMPs) at a rate of approximately 10−7 and 4 × 10−4 Mph1 helicase in vitro (36). (10 000 per 12.5 Mbp of yeast genome), respectively (3– On the other hand, studies have also implicated a 6). Removal of mis-incorporated dNMPs and rNMPs dur- role of the Smc5/6 complex during an unperturbed S ing replication requires concerted actions by distinct pro- phase (without replication stress). First, transcription of tein complexes in the mismatch repair (MMR) and ribonu- SMC6 peaks in late-G1 phase and chromosome loading
*To whom correspondence should be addressed. Tel: +1 315 464 8701; Fax: +1 315 464 8750; Email: [email protected]