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  1. Reviewer #3 (Public Review):

    The manuscript titled "The Shu complex prevents mutagenesis and cytotoxicity of single-strand specific alkylation lesions" investigates the biological function of the Shu complex in S. cerevisiae. The Shu complex, containing a DNA binding module comprised of the Csm2-Psy3 heterodimer, is conserved from budding yeast to man, and contributes to the defense against DNA damage caused by DNA alkylation. DNA alkylation occurs due to spontaneous reactions with metabolites and can be greatly increased by exogenous exposure to DNA alkylating agents. Therefore, it is an important question for how the Shu complex acts to detect and direct repair of alkylation damage. It has been well established that loss of the Shu complex sensitizes cells to alkylation damage, but the mechanism by which this complex locates sites of DNA damage and directs repair is not fully understood. This paper measures the methylation-induced mutation spectrum and uses genetic interactions to argue that the Shu complex may be involved in detecting and directing error-free repair of 3-methyl cytosine. This is a plausible hypothesis based on the body of previous work, however the evidence that Csm2-Psy3 directly detects 3-methyl cytosine sites is indirect. It would be highly significant if this complex recognizes many different structures, but future structural information is needed to understand how this could be possible.

    The strengths of the paper are in the use of whole genome sequencing to map mutation type and location in different genetic backgrounds and in the systematic testing for genetic interactions between csm2 and other DNA repair factors. It appears that the mutation spectra are very similar in the presence and absence of csm2, which suggests a broad role of the Shu complex in the cellular response to MMS.

    The impact of the work is that it could help to explain the cellular program for protection against DNA alkylating agents in budding yeast which has been a very valuable model eukaryotic organism, and raise new questions about how DNA alkylation repair pathways might function in humans that differ from yeast in important features such as in the presence of a direct repair pathway performed by ALKBH2 and ALKBH3.

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  2. Reviewer #2 (Public Review):

    The manuscript entitled "The Shu Complex Prevents Mutagenesis and Cytotoxicity of Single-Strand Specific Alkylation Lesions" by Bonilla and colleagues reports that the yeast Shu complex promotes repair of 3meC in single-stranded DNA during S phase. Specifically, the authors show that mutations and cell lethality induced by MMS in csm2∆ cells are suppressed by overexpression of the human ALKBH2. Further, the authors find that the Csm2-Psy3 module of the Shu complex has increased affinity for 3meC-containing DNA relative to unmodified DNA. The authors propose a model, where the Shu complex binds to 3meC-containing DNA to facilitate HR-dependent post-replicative gap-filling.

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  3. Reviewer #1 (Public Review):

    This study shows that the Shu complex is critical for 3meC damage tolerance in yeast, supporting the existence of a new pathway for the removal of an important DNA lesion that seems essential in yeast but likely contributes in other organisms. At the same time, it contributes to clarify the distinctive role of homologous recombination in double strand break repair and post-replicative repair.

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  4. Evaluation Summary:

    This paper is of potential interest to an audience of DNA repair and cancer biologists because it seeks to refine the mechanism by which cells respond to DNA damage. By combining a number of genetic experiments based on cell survival of different mutant combinations and mutation analysis, their results support the view that Shu is critical for 3meC damage tolerance in yeast. Notably, expression of human ALKBH2, responsible for the repair of 3meC rescues the MMS-sensitivity of Shu mutants but not that of homologous recombination mutants. The study supports the existence of a new pathway for the removal of an important DNA lesion that seems essential in yeast, but likely contributes in other organisms, and helps clarify the distinctive role of homologous recombination in DSB repair and post-replicative repair. A few additional experiments are suggested to strengthen the mechanistic conclusions and better support the central model.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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