Abstract
Helicase-like transcription factor (HLTF) and SNF2, histone-linker, PHD and RING finger domain-containing helicase (SHPRH), the two human homologs of yeast Rad5, are believed to have a vital role in DNA damage tolerance (DDT). Here we show that HLTF, SHPRH and HLTF/SHPRH knockout cell lines show different sensitivities towards UV-irradiation, methyl methanesulfonate (MMS), cisplatin and mitomycin C (MMC), which are drugs that induce different types of DNA lesions. In general, the HLTF/SHPRH double knockout cell line was less sensitive than the single knockouts in response to all drugs, and interestingly, especially to MMS and cisplatin. Using the SupF assay, we detected an increase in the mutation frequency in HLTF knockout cells both after UV- and MMS-induced DNA lesions, while we detected a decrease in mutation frequency over UV lesions in the HLTF/SHPRH double knockout cells. No change in the mutation frequency was detected in the HLTF/SHPRH double knockout cell line after MMS treatment, even though these cells were more resistant to MMS and grew faster than the other cell lines after treatment with DNA damaging agents. This phenotype could possibly be explained by a reduced activation of checkpoint kinase 2 (CHK2) and MCM2 (a component of the pre-replication complex) after MMS treatment in cells lacking SHPRH. Our data reveal both distinct and common roles of the human RAD5 homologs dependent on the nature of DNA lesions, and identified SHPRH as a regulator of CHK2, a central player in DNA damage response.
Highlights
Faithful replication of the genome is essential for life
We wanted to explore the roles of Helicase-like transcription factor (HLTF) and SHPRH in DNA damage tolerance (DDT) by using drugs which induce different types of DNA lesions
The knockout cell lines were exposed to various DNA damaging agents, after the absence of HLTF and SHPRH expression in these cell lines was verified by western blot analysis (Appendix A, Figure A1), and treatments that gave 60–70% reduction in viability on day 3 in the parental cell line were established (Appendix A, Figure A2)
Summary
Faithful replication of the genome is essential for life. DNA lesions that are not repaired prior to replication can stall replication, which may lead to mutations, replication fork collapse and genome instability. Consequences of DNA repair deficiencies are for instance carcinogenesis or neurological problems, as illustrated by the diseases Xeroderma Pigmentosum (XP), Ataxia Telangiectasia (AT) or Fanconi Anemia (FA). Cells can tolerate lesions by activating so-called DNA damage tolerance (DDT) pathways; error-prone translesion synthesis (TLS) or error-free template switch (TS) pathways after fork reversal or by strand invasion. These pathways ensure replication fork progression or restart upon replication stalling and promote the completion of DNA replication [4,5,6]
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