Genomic DNA is particularly vulnerable to mutation during S-phase when the two strands of parental duplex DNA are separated during the process of semi-conservative DNA replication. Lesions that are normally repaired efficiently in the context of double stranded DNA can cause replication forks to stall or, more dangerously, collapse. Cells from Bloom's syndrome patients, that lack the RecQ helicase BLM, show defects in the response to replicative stress and contain a multitude of chromosomal aberrations, which primarily arise through excessive levels of homologous recombination. Here, recent findings are reviewed that further our understanding of the role that BLM plays in the management of damaged replication forks.
"RecQ helicases are members of the ATP-dependent helicase family and play important roles in maintaining genome stability in eukaryotic cells during DNA repair
[1,2], DNA replication
[3,4], telomere maintenance
, and homologous recombination (HR) processes
[6,7]. Yeast has a single RecQ gene, Sgs1, but mammals and plants have multiple RecQ genes, humans having five and Arabidopsis seven
[Show abstract][Hide abstract] ABSTRACT: Background
Mammalian BLM helicase is involved in DNA replication, DNA repair and homologous recombination (HR). These DNA transactions are associated tightly with cell division and are important for maintaining genome stability. However, unlike in mammals, cell division in higher plants is restricted mainly to the meristem, thus genome maintenance at the meristem is critical. The counterpart of BLM in Arabidopsis (AtRecQ4A) has been identified and its role in HR and in the response to DNA damage has been confirmed. However, the function of AtRecQ4A in the meristem during replication stress has not yet been well elucidated.
We isolated the BLM counterpart gene OsRecQl4 from rice and analyzed its function using a reverse genetics approach. Osrecql4 mutant plants showed hypersensitivity to DNA damaging agents and enhanced frequency of HR compared to wild-type (WT) plants. We further analyzed the effect of aphidicolin—an inhibitor of S-phase progression via its inhibitory effect on DNA polymerases—on genome stability in the root meristem in osrecql4 mutant plants and corresponding WT plants. The following effects were observed upon aphidicolin treatment: a) comet assay showed induction of DNA double-strand breaks (DSBs) in mutant plants, b) TUNEL assay showed enhanced DNA breaks at the root meristem in mutant plants, c) a recombination reporter showed enhanced HR frequency in mutant calli, d) propidium iodide (PI) staining of root tips revealed an increased incidence of cell death in the meristem of mutant plants.
These results demonstrate that the aphidicolin-sensitive phenotype of osrecql4 mutants was in part due to induced DSBs and cell death, and that OsRecQl4 plays an important role as a caretaker, maintaining genome stability during DNA replication stress in the rice meristem.
"One hallmark of BS cells is a greatly elevated frequency of sister chromatid exchanges (SCE)  . Numerous studies of the role played by BLM helicase in maintaining genome stability have collectively revealed multiple roles for BLM in HR regulation       . "
[Show abstract][Hide abstract] ABSTRACT: Mutation of BLM helicase causes Blooms syndrome, a disorder associated with genome instability, high levels of sister chromatid exchanges, and cancer predisposition. To study the influence of BLM on double-strand break (DSB) repair in human chromosomes, we stably transfected a normal human cell line with a DNA substrate that contained a thymidine kinase (tk)-neo fusion gene disrupted by the recognition site for endonuclease I-SceI. The substrate also contained a closely linked functional tk gene to serve as a recombination partner for the tk-neo fusion gene. We derived two cell lines each containing a single integrated copy of the DNA substrate. In these cell lines, a DSB was introduced within the tk-neo fusion gene by expression of I-SceI. DSB repair events that occurred via homologous recombination (HR) or nonhomologous end-joining (NHEJ) were recovered by selection for G418-resistant clones. DSB repair was examined under conditions of either normal BLM expression or reduced BLM expression brought about by RNA interference. We report that BLM knockdown in both cell lines specifically increased the frequency of HR events that produced deletions by crossovers or single-strand annealing while leaving the frequency of gene conversions unchanged or reduced. We observed no change in the accuracy of individual HR events and no substantial alteration of the nature of individual NHEJ events when BLM expression was reduced. Our work provides the first direct evidence that BLM influences DSB repair pathway choice in human chromosomes and suggests that BLM deficiency can engender genomic instability by provoking an increased frequency of HR events of a potentially deleterious nature.
DNA repair 02/2011; 10(4):416-26. DOI:10.1016/j.dnarep.2011.01.009 · 3.11 Impact Factor
"BLM inhibits the D-loop formation catalyzed by RAD51, by displacing RAD51 from single-stranded DNA, thereby disrupting nucleoprotein filaments . Several models of the maintenance of genome integrity by BLM during DNA replication have been developed, most suggesting that BLM restarts replication after the stalling of the fork  . In the absence of BLM, cells display a slowing of replication fork progression associated with constitutive replication stress . "
[Show abstract][Hide abstract] ABSTRACT: Bloom's syndrome (BS) displays one of the strongest known correlations between chromosomal instability and a high risk of cancer at an early age. BS cells combine a reduced average fork velocity with constitutive endogenous replication stress. However, the response of BS cells to replication stress induced by hydroxyurea (HU), which strongly slows the progression of replication forks, remains unclear due to publication of conflicting results. Using two different cellular models of BS, we showed that BLM deficiency is not associated with sensitivity to HU, in terms of clonogenic survival, DSB generation, and SCE induction. We suggest that surviving BLM-deficient cells are selected on the basis of their ability to deal with an endogenous replication stress induced by replication fork slowing, resulting in insensitivity to HU-induced replication stress.
Journal of nucleic acids 09/2010; 2010. DOI:10.4061/2010/319754
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