The Shu complex prevents mutagenesis and cytotoxicity of single-strand specific alkylation lesions.

Braulio Bonilla,Steven A Roberts,Ewa P Malc,Alexander J Brown,Tony M Mertz,Thong T Luong,Sarah R Hengel,Catherine A Pressimone,Nima Mosammaparast,Kara A Bernstein,Adeola A Fagunloye,Piotr A Mieczkowski,Reagan A Russell,Kyle S Rapchak,Hani S Zaher,Debra Mitchell,Rudri K Vyas

doi:10.7554/elife.68080

Abstract

Three-methyl cytosine (3meC) are toxic DNA lesions, blocking base pairing. Bacteria and humans express members of the AlkB enzymes family, which directly remove 3meC. However, other organisms, including budding yeast, lack this class of enzymes. It remains an unanswered evolutionary question as to how yeast repairs 3meC, particularly in single-stranded DNA. The yeast Shu complex, a conserved homologous recombination factor, aids in preventing replication-associated mutagenesis from DNA base damaging agents such as methyl methanesulfonate (MMS). We found that MMS-treated Shu complex-deficient cells exhibit a genome-wide increase in A:T and G:C substitutions mutations. The G:C substitutions displayed transcriptional and replicational asymmetries consistent with mutations resulting from 3meC. Ectopic expression of a human AlkB homolog in Shu-deficient yeast rescues MMS-induced growth defects and increased mutagenesis. Thus, our work identifies a novel homologous recombination-based mechanism mediated by the Shu complex for coping with alkylation adducts.

Highlights

Alkylating agents such as methyl methanesulfonate (MMS) induce a diverse set of base lesions that are recognized and repaired by the base excision repair (BER) pathway or direct repair enzymes such as the AlkB family (Fu, Calvo et al, 2012, Yi & He, 2013)
Most substitutions at A:T bases in MMS-treated WT yeast A to G and A to T (Figure 1B). Previous analyses of MMS-treated mag1∆ yeast revealed that deletion of the glycosylase responsible for initiating base excision repair of 3meA resulted in a mutation spectrum predominantly composed of A to G and A to T substitutions (Mao, Brown et al, 2017), indicating that these two mutation types are likely directly induced by 3meA as opposed to 3meA-derived AP sites
The mutation spectrum from mag1∆ csm2Δ double mutant yeast mirrors that from mag1∆ yeast despite a previously determined higher MMS-induced mutation rate for mag1∆ csm2Δ than mag1-deficient yeast (Godin et al, 2016c). This spectrum, indicates that elevated frequency of A:T substitutions in mag1∆ csm2Δ yeast compared to csm2Δ yeast is due to higher amounts of 3meA present that are normally bypassed with the help of the Shu complex. 7meG is the most common MMS146 induced DNA adduct at G:C base pairs and is not itself mutagenic (Shrivastav et al, 2010)

Summary

Introduction

Alkylating agents such as methyl methanesulfonate (MMS) induce a diverse set of base lesions that are recognized and repaired by the base excision repair (BER) pathway or direct repair enzymes such as the AlkB family (Fu, Calvo et al, 2012, Yi & He, 2013) When these lesions, or its repair intermediates, are encountered by a replisome, replication fork stalling can occur (Shrivastav, Li et al, 2010, Sobol, Kartalou et al, 2003). In this scenario, DNA base damage is preferentially bypassed using homologous recombination (HR) or translesion synthesis (TLS), postponing its repair but allowing replication to be completed in a timely fashion. TLS serves as an alternative pathway to error-free lesion bypass, as disruption of genes involved in the error-free PRR pathway leads to an increase in mutations that is dependent on TLS (Broomfield, Chow et al, 1998, Huang, Rio et al, 2003, Swanson, Morey et al, 1999)

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Conclusion