The Human RAD5 Homologs, HLTF and SHPRH, Have Separate Functions in DNA Damage Tolerance Dependent on the DNA Lesion Type

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.

A role for Rad5 in ribonucleoside monophosphate (rNMP) tolerance

“…HLTF is more similar to Rad5 in regards to sequence homology and structural resemblance in which HLTF but not SHPRH contains the HIRAN domain which recognizes 3′ssDNA and performs DNA-dependent ATPase-catalyzing fork reversal upon DNA damage ( Chavez et al, 2018 ). Rad5 homologs HLTF and SHPRH have distinct roles in mediating DDT in response to different DNA damaging agents rather than acting redundantly ( Seelinger et al, 2020 ). In response to MMS-induced DNA lesions, HLTF regulates error-free DDT pathway via promoting TS with its Ub ligase domain or promoting fork regression with its ATP-dependent translocase activity and HIRAN domain.…”

“…HLTF can also regulate TLS via Pol η which bypasses MMS-induced lesions in a relatively accurate manner, thus reducing the mutagenesis rate after MMS treatment. On the other hand, SHPRH repairs MMS-induced DNA damage by preventing double strand breaks and regulation of checkpoint activation rather than a role in TLS ( Seelinger et al, 2020 ).…”

“…Rad5 human orthologs: a potential role in rNMP bypass HLTF and SHPRH are also E3 ubiquitin ligases that poly-ubiquitinate PCNA to direct DTT towards TS. Similar to their yeast homologue Rad5, they interact with Rad18 and Mms2-Ubc13 complexes to polyubiquitinate PCNA triggering the template switching (TS) pathway (Seelinger et al, 2020). HLTF is more similar to Rad5 in regards to sequence homology and structural resemblance in which HLTF but not SHPRH contains the HIRAN domain which recognizes 39ssDNA and performs DNA-dependent ATPase-catalyzing fork reversal upon DNA damage (Chavez et al, 2018).…”

Rad5 and Its Human Homologs, HLTF and SHPRH, Are Novel Interactors of Mismatch Repair

“…Both HLTF and SHPRH have E3 ubiquitin ligase ability, both can polyubiquitinate PCNA, and HLTF can complement UV sensitivity of a rad5Δ S. cerevisiae strain ( Unk et al, 2006 ; Unk et al, 2008 ; Masuda et al, 2012 ). HLTF and SHPRH also have direct but distinct roles in directing TLS- and TS-mediated PRR, and HLTF and SHPRH deletion mutants have different sensitivities to agents that cause DNA lesions ( Seelinger et al, 2020 ). HLTF enhances TLS and inhibits SHPRH following UV damage, but MMS treatment instead causes SHPRH response and HLTF degradation ( Lin et al, 2011 ).…”

DNA Damage Response

“…Seelinger et al [ 17 ] generated and characterized single-knockout human cell lines lacking helicase-like transcription factor (HLTF), SNF2, histone-linker, PHD, and RING finger domain-containing helicase (SHPRH), and a double-knockout HLTF/SHPRH. Various DNA damage types were introduced in these cells using UV light, methyl methanesulfonate (MMS), mitomycin C (MMC), and cisplatin.…”