Abstract
Defects in the ability to respond properly to an unrepaired DNA lesion blocking replication promote genomic instability and cancer. Human HLTF, implicated in error-free replication of damaged DNA and tumour suppression, exhibits a HIRAN domain, a RING domain, and a SWI/SNF domain facilitating DNA-binding, PCNA-polyubiquitin-ligase, and dsDNA-translocase activities, respectively. Here, we investigate the mechanism of HLTF action with emphasis on its HIRAN domain. We found that in cells HLTF promotes the filling-in of gaps left opposite damaged DNA during replication, and this postreplication repair function depends on its HIRAN domain. Our biochemical assays show that HIRAN domain mutant HLTF proteins retain their ubiquitin ligase, ATPase and dsDNA translocase activities but are impaired in binding to a model replication fork. These data and our structural study indicate that the HIRAN domain recruits HLTF to a stalled replication fork, and it also provides the direction for the movement of the dsDNA translocase motor domain for fork reversal. In more general terms, we suggest functional similarities between the HIRAN, the OB, the HARP2, and other domains found in certain motor proteins, which may explain why only a subset of DNA translocases can carry out fork reversal.
Highlights
Unrepaired DNA lesions can block the movement of the replication machinery leading to DNA strand breaks and chromosomal rearrangements providing eventually the driving force to cancer [1]
Alignment of the HIRAN domains present in various proteins (Figure 1B) revealed its highly conserved residues, and our model indicated that the HIRAN domain is a sixstranded -barrel covered with a single ␣-helix at one end and either a short helix or a loop at the other end
We report that HLTF plays a critical role in filling in the gaps remaining opposite the damaged DNA after the majority of DNA has been replicated, but its depletion does not slow down the overall rate of DNA maturation during the replication of undamaged DNA
Summary
Unrepaired DNA lesions can block the movement of the replication machinery leading to DNA strand breaks and chromosomal rearrangements providing eventually the driving force to cancer [1]. To minimize these consequences, cells possess distinct mechanisms for replicating through damaged DNA such as translesion synthesis or template switching [2,3,4]. Not all types of lesions in the template strand can be accommodated into the active site of TLS polymerases, and they cannot provide a general solution for replication through all types of DNA damage [2,7]. To generate the four-stranded chicken foot, both the leading and the lagging nascent strands at the replication fork should be displaced
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