The DNA repair protein SHPRH is a nucleosome-stimulated ATPase and a nucleosome-E3 ubiquitin ligase

Abstract

Background Maintenance of genome integrity during DNA replication is crucial to the perpetuation of all organisms. In eukaryotes, the bypass of DNA lesions by the replication machinery prevents prolonged stalling of the replication fork, which could otherwise lead to greater damages such as gross chromosomal rearrangements. Bypassing DNA lesions and subsequent repair are accomplished by the activation of DNA damage tolerance pathways such as the template switching (TS) pathway. In yeast, the RAD5 (Radiation-sensitive 5) protein plays a crucial role in initiating the TS pathway by catalyzing the polyubiquitination of PCNA (Proliferation Cell Nuclear Antigen). Likewise, one of the mammalian RAD5-homologs, SHPRH (SNF2, histone linker, PHD, RING, helicase) mediates PCNA polyubiquitination. To date, the study of SHPRH enzymatic functions has been limited to this modification. It is therefore unclear how SHPRH carries out its function in DNA repair. Moreover, how this protein regulates gene transcription at the enzymatic level is also unknown. Results Given that SHPRH harbors domains found in chromatin remodeling proteins, we investigated its biochemical properties in the presence of nucleosomal substrates. We find that SHPRH binds equally well to double-stranded (ds) DNA and to nucleosome core particles, however, like ISWI and CHD-family remodelers, SHPRH shows a strong preference for nucleosomes presenting extranucleosomal DNA. Moreover, nucleosomes but not dsDNA strongly stimulate the ATPase activity of SHPRH. Intriguingly, unlike typically observed with SNF2-family enzymes, ATPase activity does not translate into conventional nucleosome remodeling, under standard assay conditions. To test whether SHPRH can act as a ubiquitin E3 ligase for nucleosomes, we performed a screen using 26 E2-conjugating enzymes. We uncover that SHPRH is a potent nucleosome E3-ubiquitin-ligase that can function with at least 7 different E2s. Mass spectrometry analyses of products generated in the presence of the UBE2D1-conjugating enzyme reveal that SHPRH can catalyze the formation of polyubiquitin linkages that are either branched or associated with the recruitment of DNA repair factors, as well as linkages involved in proteasomal degradation. Conclusions We propose that, in addition to polyubiquitinating PCNA, SHPRH promotes DNA repair or transcriptional regulation in part through chromatin ubiquitination. Our study sets a biochemical framework for studying other RAD5- and RAD16-related protein functions through the ubiquitination of nucleosomes. Electronic supplementary material The online version of this article (10.1186/s13072-019-0294-5) contains supplementary material, which is available to authorized users.

Overarching control of autophagy and DNA damage response by CHD6 revealed by modeling a rare human pathology

“…2a ), and with the fact that the HSS mutation is not at a lysine or arginine residue, both of which were shown to be important for DNA binding by SANT-SLIDE modules 7 . To exclude that additional DNA binding modules in CHD6, like its chromodomains 48 , 49 , might compensate for the I1600M mutation, we purified and tested the second putative CHD6 SANT-SLIDE module alone in EMSAs. Again, wild-type and mutant CHD6 SANT-SLIDE modules did bind DNA and nucleosomes with similar efficiencies (Supplementary Fig.…”

“…Steps from cloning to protein expression and purification were carried out as described previously 48 . In brief, WT and mutant I1600M CHD6-HA cDNAs were cloned into pFastBac1 and the HA tag was replaced by a FLAG tag using PCR and Gibson assembly.…”

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

“…How SHPRH knockdown reduces CHK2 activation is elusive. The reduced CHK2 activation could possibly be caused by lack of the ubiquitin ligase activity of SHPRH and increased stability of other ubiquitin ligases responsible for CHK2 turnover e.g., via SIAH2 [36], or by the recently suggested role of SHPRH as a nucleosome-E3 ubiquitin ligase, which could possibly change the gene expression level of CHK2 [37]. The ubiquitin ligase activity of SHPRH could also regulate degradation of proteins involved in activation of origin firing leading to increased origin firing independent of CHK2.…”

Overarching control of autophagy and DNA damage response by CHD6 revealed by modeling a rare human pathology

“…Steps from cloning to protein expression and purification were carried out as described previously 80 .…”

“…All baculoviruses were produced according to the Bac-to-Bac Baculovirus Expression Systems manual (Life Technologies) and CHD6-FLAG protein constructs were next purified from ~4 L of Sf9 insect cell cultures. Purified proteins were used in electrophoretic mobility shift assays (EMSA), as well as in nucleosome mobilization/sliding and restriction enzyme accessibility (REA) assays using DNA and nucleosome substrates as described previously 80 . Detection and quantification of non-radioactive substrates was achieved following staining with SYBR Gold Nucleic Acid Gel Stain (ThermoFisher).…”