Role of Double-Stranded DNA Translocase Activity of Human HLTF in Replication of Damaged DNA

Abstract

Unrepaired DNA lesions can block the progression of the replication fork, leading to genomic instability and cancer in higher-order eukaryotes. In Saccharomyces cerevisiae, replication through DNA lesions can be mediated by translesion synthesis DNA polymerases, leading to error-free or error-prone damage bypass, or by Rad5-mediated template switching to the sister chromatid that is inherently error free. While translesion synthesis pathways are highly conserved from yeast to humans, very little is known of a Rad5-like pathway in human cells. Here we show that a human homologue of Rad5, HLTF, can facilitate fork regression and has a role in replication of damaged DNA. We found that HLTF is able to reverse model replication forks, a process which depends on its double-stranded DNA translocase activity. Furthermore, from analysis of isolated dually labeled chromosomal fibers, we demonstrate that in vivo, HLTF promotes the restart of replication forks blocked at DNA lesions. These findings suggest that HLTF can promote error-free replication of damaged DNA and support a role for HLTF in preventing mutagenesis and carcinogenesis, providing thereby for its potential tumor suppressor role.

Supplemental material for this article may be found at http://mcb.asm.org/.

We thank Katalin Kovács and Ildikó Kravjar for the technical assistance, Jung-Hoon Yoon for optimizing siRNA knockdown of HLTF, Barnabas Szakal for helping in siRNA experiments and generating the GST-FLAG-HLTF-overexpressing plasmid, and Dean A. Jackson for sharing his expertise on fiber assays.

This work was supported by the Howard Hughes Medical Institute grant 55005612, the Wellcome Trust International Senior Research Fellowship, Hungarian Science Foundation grants OTKA 77495 and TÁMOP-4.2.2/08/1, the János Bolyai Research Scholarship of the Hungarian Academy of Sciences, the Hungarian National Office for Research and Technology grant KFKT-1-2006-0010, and the FEBS Collaborative Experimental Scholarships for Central & Eastern Europe.

We declare no competing financial interests.