Human HEL308 localizes to damaged replication forks and unwinds lagging strand structures.

HEL308 is a superfamily II DNA helicase, conserved from archaea through to humans. HEL308 family members were originally isolated by their similarity to the Drosophila melanogaster Mus308 protein, which contributes to the repair of replication-blocking lesions such as DNA interstrand cross-links. Biochemical studies have established that human HEL308 is an ATP-dependent enzyme that unwinds DNA with a 3' to 5' polarity, but little else is know about its mechanism. Here, we show that GFP-tagged HEL308 localizes to replication forks following camptothecin treatment. Moreover, HEL308 colocalizes with two factors involved in the repair of damaged forks by homologous recombination, Rad51 and FANCD2. Purified HEL308 requires a 3' single-stranded DNA region to load and unwind duplex DNA structures. When incubated with substrates that model stalled replication forks, HEL308 preferentially unwinds the parental strands of a structure that models a fork with a nascent lagging strand, and the unwinding action of HEL308 is specifically stimulated by human replication protein A. Finally, we show that HEL308 appears to target and unwind from the junction between single-stranded to double-stranded DNA on model fork structures. Together, our results suggest that one role for HEL308 at sites of blocked replication might be to open up the parental strands to facilitate the loading of subsequent factors required for replication restart.

Differential contributions of mammalian Rad54 paralogs to recombination, DNA damage repair, and meiosis.

Homologous recombination is a versatile DNA damage repair pathway requiring Rad51 and Rad54. Here we show that a mammalian Rad54 paralog, Rad54B, displays physical and functional interactions with Rad51 and DNA that are similar to those of Rad54. While ablation of Rad54 in mouse embryonic stem (ES) cells leads to a mild reduction in homologous recombination efficiency, the absence of Rad54B has little effect. However, the absence of both Rad54 and Rad54B dramatically reduces homologous recombination efficiency. Furthermore, we show that Rad54B protects ES cells from ionizing radiation and the interstrand DNA cross-linking agent mitomycin C. Interestingly, at the ES cell level the paralogs do not display an additive or synergic interaction with respect to mitomycin C sensitivity, yet animals lacking both Rad54 and Rad54B are dramatically sensitized to mitomycin C compared to either single mutant. This suggests that the paralogs possibly function in a tissue-specific manner. Finally, we show that Rad54, but not Rad54B, is needed for a normal distribution of Rad51 on meiotic chromosomes. Thus, even though the paralogs have similar biochemical properties, genetic analysis in mice uncovered their nonoverlapping roles.

DNA double-strand break repair and chromosome translocations.

Translocations are genetic aberrations that occur when a broken fragment of a chromosome is erroneously rejoined to another chromosome. The initial event in the creation of a translocation is the formation of a DNA double-strand break (DSB), which can be induced both under physiological situations, such as during the development of the immune system, or by exogenous DNA damaging agents. Two major repair pathways exist in cells that repair DSBs as they arise, namely homologous recombination, and non-homologous end-joining. In some situations these pathways can function inappropriately and rejoin ends incorrectly to produce genomic rearrangements, including translocations. Translocations have been implicated in cancer because of their ability to activate oncogenes. Due to selection at the level of the DNA, the cell, and the tissue certain forms of cancer are associated with specific translocations that can be used as a tool for diagnosis and prognosis of these cancers.

The response of mammalian cells to UV-light reveals Rad54-dependent and independent pathways of homologous recombination.

Ultraviolet (UV) radiation-induced DNA lesions can be efficiently repaired by nucleotide excision repair (NER). However, NER is less effective during replication of UV-damaged chromosomes. In contrast, translesion DNA synthesis (TLS) and homologous recombination (HR) are capable of dealing with lesions in replicating DNA. The core HR protein in mammalian cells is the strand exchange protein RAD51, which is aided by numerous proteins, including RAD54. We used RAD54 as a cellular marker for HR to study the response of mammalian embryonic stem (ES) cells to UV irradiation. In contrast to yeast, ES cells lacking RAD54 are not UV sensitive. Here we show that the requirement for mammalian RAD54 is masked by active NER. By genetically inactivating NER and HR through disruption of the Xpa and Rad54 genes, respectively, we demonstrate the contribution of HR to chromosomal integrity upon UV irradiation. We demonstrate using chromosome fiber analysis at the individual replication fork level, that HR activity is important for the restart of DNA replication after induction of DNA damage by UV-light in NER-deficient cells. Furthermore, our data reveal RAD54-dependent and -independent contributions of HR to the cellular sensitivity to UV-light, and they uncover that RAD54 can compensate for the loss of TLS polymerase η with regard to UV-light sensitivity. In conclusion, we show that HR is important for the progression of UV-stalled replication forks in ES cells, and that protection of the fork is an interplay between HR and TLS.