Although it is not part of the normal replication machinery, BRCA2 localizes in the nuclear foci that are formed when replication is inhibited. So, does BRCA2 function in the cellular response to stalled replication? Venkitaraman and colleagues investigated this by blocking DNA synthesis in mouse cells that were homozygous for a targeted truncation of Brca2 — Brca2Tr — that produces a non-functional protein. They used two-dimensional gel electrophoresis to visualize Y-arcs — DNA structures that are formed at replication forks — at the ribosomal DNA (rDNA) locus. When replication was inhibited using hydroxyurea (HU), these structures disappeared from Brca2Tr/Tr cells, but persisted in wild-type cells, indicating that Brca2 is required for the stability of stalled replication forks.
In replication-defective bacteria, stalled replication forks can be broken down into linear chromosomal fragments. If a similar process occurred as a result of loss of Brca2 function, this could explain the spontaneous genomic instability that arises as a result of mutations in this gene. To investigate this, the authors used pulsed-field gel electrophoresis (PFGE) to check for the production of DNA fragments that would be predicted to form because of the breakdown of replication forks in the rDNA locus. Chromosomal DNA from this locus is usually too large to be visualized by PFGE, but in Brca2Tr/Tr cells, fragments consistent with breakdown at rDNA replication forks were seen following application of HU. This effect of Brca2 inactivation is not restricted to the rDNA genes, as similar breakdown events were shown to occur at other genomic loci.
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