Homologous recombination is associated with the dynamic assembly and disassembly of DNA-protein complexes. Assembly of a nucleoprotein filament comprising ssDNA and the RecA homolog, Rad51, is a key step required for homology search during recombination. The budding yeast Srs2 DNA translocase is known to dismantle Rad51 filament in vitro. However, there is limited evidence to support the dismantling activity of Srs2 in vivo. Here, we show that Srs2 indeed disrupts Rad51-containing complexes from chromosomes during meiosis. Overexpression of Srs2 during the meiotic prophase impairs meiotic recombination and removes Rad51 from meiotic chromosomes. This dismantling activity is specific for Rad51, as Srs2 Overexpression does not remove Dmc1 (a meiosis-specific Rad51 homolog), Rad52 (a Rad51 mediator), or replication protein A (RPA; a single-stranded DNA-binding protein). Rather, RPA replaces Rad51 under these conditions. A mutant Srs2 lacking helicase activity cannot remove Rad51 from meiotic chromosomes. Interestingly, the Rad51-binding domain of Srs2, which is critical for Rad51-dismantling activity in vitro, is not essential for this activity in vivo. Our results suggest that a precise level of Srs2, in the form of the Srs2 translocase, is required to appropriately regulate the Rad51 nucleoprotein filament dynamics during meiosis. RECOMBINATION between homologous sequences promotes genomic stability. As such, a wide variety of DNAassociated proteins and protein complexes regulate the recombination process. Within chromatin, the chromosome structure probably also directly affects the assembly and disassembly of protein complexes involved in recombination. To understand the molecular mechanisms that govern assembly and disassembly of these complexes, homologous recombination during meiosis can be used as a model system.Meiotic recombination is initiated when Spo11, a meiosisspecific topoisomerase-like protein, generates double-strand breaks (DSBs) (Keeney 2001). The 59 ends of these DSBs are quickly resected to produce a 39-overhanging stretch of single-stranded DNA (ssDNA). The ssDNA is used to search for homologous double-stranded DNAs (dsDNAs). Once homology between the ss-and dsDNAs are matched, the ssDNA is invaded into the dsDNA. Strand invasion leads to DNA synthesis with the 39 end of the invading strand as the primer. The resulting intermediate is then converted into the singleinvasion intermediate (SEI) and then a DNA structure with double Holiday junctions (dHJ) (Schwacha and Kleckner 1994;Hunter and Kleckner 2001). Resolution of dHJ structures typically results in chromosomal crossover (CO) (Allers and Lichten 2001;Hunter and Kleckner 2001). Alternatively, newly synthesized DNA of the invading strand after the displacement can anneal with ssDNA from the other end of the DSB, which results in the formation of a noncrossover (NCO) product (through the synthesis-dependent strand-annealing pathway). In meiosis, the recombination occurs preferentially between homologous chromosomes rather than betw...
Proper repair of double-strand breaks (DSBs) is key to ensure proper chromosome segregation. In this study, we found that the deletion of the SRS2 gene, which encodes a DNA helicase necessary for the control of homologous recombination, induces aberrant chromosome segregation during budding yeast meiosis. This abnormal chromosome segregation in srs2 cells accompanies the formation of a novel DNA damage induced during late meiotic prophase-I. The damage may contain long stretches of single-stranded DNAs (ssDNAs), which lead to aggregate formation of a ssDNA binding protein, RPA, and a RecA homolog, Rad51, as well as other recombination proteins inside of the nuclei. The Rad51 aggregate formation in the srs2 mutant depends on the initiation of meiotic recombination and occurs in the absence of chromosome segregation. Importantly, as an early recombination intermediate, we detected a thin bridge of Rad51 between two Rad51 foci or among the foci in the srs2 mutant, which is rarely seen in wild type. These might be cytological manifestation of the connection of two DSB ends and multi-invasion. The DNA damage with Rad51 aggregates in the srs2 mutant is passed through anaphase-I and -II, suggesting the absence of DNA damage-induced cell-cycle arrest after the pachytene stage.We propose that Srs2 helicase resolves early protein-DNA recombination intermediates to suppress the formation of aberrant lethal DNA damage during late prophase-I.
Bacteria have a variety of resistance mechanisms for surviving bacteriophage infections. Here, we describe a novel anti-phage mechanism in Escherichia coli. Cells harboring a plasmid with the genes abpA and abpB, formerly yfjL and yfjK, blocked the propagation of bacteriophages belonging to three families: T4, T2, T7 and λ phages. Both genes were necessary for the inhibition of phage propagation, and deletion of either chromosomal gene resulted in a 20% increase of progeny compared to wild-type cells. Neither overexpression nor deficiency of AbpA and AbpB had any apparent effect on E. coli growth. We isolated seven suppressor mutants of T4 phage that grew weakly on cells overexpressing AbpA and AbpB, and found that their mutations were all located in gene 41, which encodes a replicative DNA helicase that is essential for DNA replication. Furthermore, we demonstrated that AbpA and AbpB inhibited DNA replication and late gene expression of T4 phage. Similarly, DNA replication of T7 and λ phages was also inhibited by AbpA and AbpB. These results strongly suggest that E. coli AbpA and AbpB target DNA replication of phages to block their propagation.
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