Homologous recombination of monkey alpha-satellite repeats in an in vitro simian virus 40 replication system: possible association of recombination with DNA replication.

Abstract

To study homologous recombination between repeated sequences in an in vitro simian virus 40 (SV40) replication system, we constructed a series of substrate DNAs that contain two identical fragments of monkey a-satellite repeats. Together with the SV40-pBR322 composite vector encoding Apr and KMr, the DNAs also contain the Escherichia coli galactokinase gene (galK) positioned between two a-satellite fragments. The a-satellite sequence used consists of multiple units of tandem 172-bp sequences which differ by microheterogeneity. The substrate DNAs were incubated in an in vitro SV40 DNA replication system and used to transform the E. coli galK strain DH1OB after digestion with DpnI. The number of E. coli galK Apr Kmr colonies which contain recombinant DNAs were determined, and their structures were analyzed. Products of equal and unequal crossovers between identical 172-bp sequences and between similar but not identical (homeologous) 172-bp sequences, respectively, were detected, although those of the equal crossover were predominant among all of the galK mutant recombinants. Similar products were also observed in the in vivo experiments with COS1 cells. The in vitro experiments showed that these recombinations were dependent on the presence of both the SV40 origin of DNA replication and SV40 large T antigen. Most of the recombinant DNAs were generated from newly synthesized DpnI-resistant DNAs. These results suggest that the homologous recombination observed in this SV40 system is associated with DNA replication and is suppressed by mismatches in heteroduplexes formed between similar but not identical sequences.It is known that the genomic DNA of higher eukaryotes, including mammals, contains large amounts of repetitive DNAs. Among them, at-satellite DNA is known to localize in centromeric regions of primate chromosomes as tandem arrays of thousands of kilobases. Homologous recombinations between these repetitive DNA sequences potentially play a crucial role in drastic chromosomal rearrangements such as chromosomal deletion, duplication, inversion, amplification, and translocation. In this context, it is surprising that such chromosomal rearrangements are usually observed very rarely in mammalian cells. The study of homologous recombination between various repetitive DNA sequences is thought to be one of the keys to understanding how chromosome integrity is maintained during the cell cycle in mammalian cells.Recent reports have shown that intrachromosomal trinucleotide repeat expansion is the cause of several human genetic diseases: myotonic dystrophy, fragile X syndrome, spinal bulbar muscular atrophy, and Huntington's disease (5, 12-14, 19, 21, 26, 31, 42). These rearrangements are thought to occur by either unequal crossing-over, i.e., crossovers between misaligned repeats, or DNA polymerase slippage, i.e., reassociation of repeats in a misaligned configuration during DNA replication. It is important to understand the mechanism of expansions or diminutions of repeats in mammalian cells.In Saccharomyces cerevis...

Wild-type p53 inhibits replication-associated homologous recombination

“…Therefore, as was observed by others for UV, irradiation during replication should lead to increased levels of chromatid breaks (Kaufmann and Wilson, 1994). Replication-associated recombination serves to bypass unrepaired DNA lesions that inhibit chain elongation, and there are hints that also in SV40 based systems homologous recombination is coupled to DNA replication (Sarasin and Hanawalt, 1978;Kawasaki et al, 1994;Cox et al, 2000). Therefore, we propose that the p53-dependent downregulation of radiation induced recombination is directed towards replication-associated processes.…”

Survey of extrachromosomal circular DNA derived from plant satellite repeats

“…Moreover, the eccDNA patterns detected for different VicTR-A and VicTR-B subfamilies or higher-order repeats suggest that relatively long regions (tens to hundreds of nucleotides) of high sequence similarity are required for efficient recombination. It has been reported that efficiency of homologous recombination depends on similarity of involved sequences [ 34 - 36 ] and is proportional to the length of the similarity [ 37 ]. If the length or degree of sequence similarity is decreased, the rate of recombination is reduced rapidly.…”

The essential role of yeast topoisomerase III in meiosis depends on recombination

“…The absence of Top3 in mitosis leads to the accumulation of cells with an undivided nucleus in the neck of an elongated bud (Gangloff et al ., 1994b). The associated cell cycle delay at the G 2 –M transition is consistent with an impediment of chromosome segregation, suggesting that Top3 is involved directly in the separation of replicated chromatids or, alternatively, in the resolution of topological structures resulting from either recombination during replication or collapsed replication forks (Young et al ., 1984; Kawasaki et al ., 1994; Zou and Rothstein, 1997). Because no noticeable difference in the timing of diploid spore formation could be detected when meiotic levels of recombination are abolished, we do not believe it likely that Top3 plays a meiotic‐specific role in the DNA synthesis process.…”