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Abstract
To prevent replication fork collapse and genome instability under replicative stress, DNA damage tolerance (DDT) mechanisms have evolved. The RAD5 homologs, HLTF (helicase-like transcription factor) and SHPRH (SNF2, histone-linker, PHD and RING finger domain-containing helicase), both ubiquitin ligases, are involved in several DDT mechanisms; DNA translesion synthesis (TLS), fork reversal/remodeling and template switch (TS). Here we show that these two human RAD5 homologs contain functional APIM PCNA interacting motifs. Our results show that both the role of HLTF in TLS in HLTF overexpressing cells, and nuclear localization of SHPRH, are dependent on interaction of HLTF and SHPRH with PCNA. Additionally, we detected multiple changes in the mutation spectra when APIM in overexpressed HLTF or SHPRH were mutated compared to overexpressed wild type proteins. In plasmids from cells overexpressing the APIM mutant version of HLTF, we observed a decrease in C to T transitions, the most common mutation caused by UV irradiation, and an increase in mutations on the transcribed strand. These results strongly suggest that direct binding of HLTF and SHPRH to PCNA is vital for their function in DDT.
Highlights
Cells are constantly exposed to endogenous and exogenous agents that cause DNA lesions
We show that AlkB homolog PCNA interacting motif (APIM) in both HLTF and SHPRH interact with PCNA, and that the binding of HLTF and SHPRH to PCNA is important for their function in DNA damage tolerance (DDT) after UV-induced DNA damage
APIM in HLTF (KFIVK) fused to CFP and co-expressed with HcRed-tagged PCNA colocalized with PCNA in foci resembling replication foci (Figure 1A, mid two images)
Summary
Cells are constantly exposed to endogenous and exogenous agents that cause DNA lesions. Human cells have at least two additional important fork reversal proteins, SMARCAL1 and ZRANB3 The latter contains a functional PCNA interacting sequence, AlkB homolog 2 PCNA interacting motif (APIM) [17]. SMARCAL1, ZRANB3, and HLTF can restore a three-way-replication fork from a four-way-reversed replication fork [16,18] Both RAD5 homologs are reported to be involved in TLS: HLTF by recruiting POLη after UV-induced DNA damage, and SHPRH by recruiting POLκ after methyl methanesulfonate (MMS)-induced DNA damage [19]. HLTF and SHPRH are regarded as candidate tumor suppressor genes, because loss of function or dysregulation has been linked to cancer development [20,21,22], and HLTF is often epigenetically silenced by promotor hypermethylation in colon cancers (~40%) [23] Both RAD5 homologs contain potential PCNA interacting motifs, APIMs, within the helicase domain at their C-terminus; KFIVK (amino acid (aa) 959–963) in HLTF and RFLIK (aa 1631–1635) in SHPRH. We show that APIM in both HLTF and SHPRH interact with PCNA, and that the binding of HLTF and SHPRH to PCNA is important for their function in DDT after UV-induced DNA damage
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