HMCES safeguards replication from oxidative stress and ensures error‐free repair

Abstract

Replication across oxidative DNA lesions can give rise to mutations that pose a threat to genome integrity. How such lesions, which escape base excision repair, get removed without error during replication remains unknown. Our PCNA‐based screen to uncover changes in replisome composition under different replication stress conditions had revealed a previously unknown PCNA‐interacting protein, HMCES/C3orf37. Here, we show that HMCES is a critical component of the replication stress response, mainly upon base misincorporation. We further demonstrate that the absence of HMCES imparts resistance to pemetrexed treatment due to error‐prone bypass of oxidative damage. Furthermore, based on genetic screening, we show that homologous recombination repair proteins, such as CtIP, BRCA2, BRCA1, and PALB2, are indispensable for the survival of HMCES KO cells. Hence, HMCES, which is the sole member of the SRAP superfamily in higher eukaryotes known so far, acts as a proofreader on replication forks, facilitates resolution of oxidative base damage, and therefore ensures faithful DNA replication.

Loss of HMCES is synthetic lethal with APOBEC activity in cancer cells

“…In addition, our secondary screens identified the mismatch repair protein MSH2 as positively selected, consistent with our recent data suggesting that APOBEC3A-mediated mutagenesis is mediated by MMR activity, resulting in the characteristic omikli pattern of clustered mutations[7]. In contrast, UNG, MSH2 or other MMR proteins, were not identified as strong influencers of the survival of HMCES KO cells in the absence of A3A or DNA damage in our screens, or in recent screens performed by others in HMCES KO cells in a HEK293 background[76].…”

“…TP53 status was shown to have a major influence on CRISPR/Cas9 survival screens and it is intuitive that checkpoint status would limit toxic DNA damage generated during S-phase and prevent mitotic catastrophe resulting from under-replicated DNA entering mitosis[78]. As A3A-mediated damage of ssDNA is S-phase specific, our results would again be consistent with the recently proposed model that HMCES shields AP-sites in ssDNA from processing by BER endonucleases that would generate AP-sites and toxic DSBs, or from replication by TLS polymerases that would result in increased mutagenesis[27,42,43,49,76]. As the enzymatic activities of HMCES have been implicated in this function, accumulating data suggests that targeting HMCES would be an attractive strategy for the specific sensitization of A3A-expressing, TP53-deficient cancers.…”

“…Aside from HMCES, the overall agreement between cell lines at the individual gene level was limited in our A3A screens. A previous analysis of essential genes using a DNA repair library in HEK293 HMCES KO cells identified numerous proteins involved in HR and TLS, suggesting that loss of HMCES was synthetically lethal with the attenuation of these repair pathways[76]. In the absence of A3A expression, we observed very limited overlap with that study when compared to our data in LXF-289 HMCES KO cells, with the ribonucleotide reductase subunit RRM1, clamp loader subunit RFC3 and HR factor XRCC3 being the only notable overlapping hits among negatively selected genes.…”

Mechanisms of damage tolerance and repair during DNA replication

“…In contrast to the above model, a recent study has shown that AP endonucleases may act in concert with HMCES for the resolution of AP sites. This study suggested that the HMCES-DPC may help to recruit AP endonuclease to channel these lesions to the proper repair pathway ( 145 ). In support of this, AP sites are known to react with several proteins such as histones, KU heterodimer, human ribosomal protein S3, nucleoside diphosphate kinase, PARP1, and several others ( 146–152 ).…”

How to fix DNA-protein crosslinks

“…For example, if the AP site was to be processed on ssDNA, it would generate a detrimental DSB. Accordingly, HMCES-deficient cells rely on HR proteins for survival and accumulate DSBs upon APOBEC3 overexpression, which stimulates the formation of abasic sites [ 41 , 105 ]. Thus, by crosslinking to the AP site, HMCES might ensure that the AP site is not processed until gap filling synthesis has been completed and the lesion is safely located on dsDNA ( Fig.…”