Short direct repeats mediate spontaneous high-frequency deletions in DNA of minute virus of mice.

Abstract

, J. Virol. 32:276-292, 1979) revealed a remarkably high rate of spontaneous deletion in viral DNA during lytic infection of cultured murine cells with minute virus of mice (MVM), an autonomous parvovirus. In the present study, we have isolated plasmid and phage recombinants containing MVM DNA inserts bearing deletions and we have determined the DNA sequence spanning three deletion junctions. The deletions, which average 3 kilobases in length, occur between pairs of perfectly homologous 4-to 10-base-pair direct repeats, such that one copy of the repeated sequence is lost, whereas the other remains behind at the deletion junction. When compared, the three sets of direct repeats exhibit no apparent sequence homology and have an A+T content of between 50 and 80%. These results indicate that 4-to 10-base-pair homologies mediate spontaneous deletion formation in the MVM genome and highlight parvoviruses as novel model systems for studies of this ubiquitous pathway of genetic variation.Spontaneous deletions account for many independent mutations within the lacd gene (1, 6), the tryptophan operon (25), and the transposable element IS1 of Escherichia coli (18) as well as the rII region of bacteriophage T4 (14) and the early region of bacteriophage T7 (20). In addition, the precise excision of certain procaryotic transposable elements such as TnJO may be viewed as analogous to spontaneous deletion formation (1, 9). In eucaryotes, spontaneous deletions, rather than base substitutions, are largely responsible for divergence between members of the 3-globin gene family in humans (5) and also have been found within integrated viral sequences present in the polyoma-transformed cell line 82-Rat (15). High-frequency mutation at the adenine phosphoribosyltransferase locus in Chinese hamster ovary cells is also due to deletion of the gene (17). In most of these cases, the deletions are associated with short, directly repeated DNA sequences that flank the deleted DNA segments, and a model that invokes slipped mispairing during DNA replication has been proposed to explain how such direct repeats might participate in the deletion process (1,5,19). We have extended these observations in the present study of high-frequency deletion mutations that arise spontaneously in the DNA genome of the parvovirus minute virus of mice (MVM) (8).MVM is a member of a large group of mammalian parvoviruses that have proven to be useful model systems for the study of mammalian DNA replication (4, 23). The MVM genome is a linear single-stranded DNA molecule consisting of 5,081 nucleotide residues with terminal self-complementary sequences that exist in the form of hairpin duplexes (3). Viral DNA replication occurs by a self-priming mechanism that leads to the accumulation of linear monomeric and concatemeric duplex viral replicative forms (2, 7) and is * Corresponding author. dependent upon cellular functions expressed transiently in the late S or early G2 phase of the cell cycle (22,24).Of the progeny virus resulting from a single high-multiplicity infe...

Nonhomologous recombination in mammalian cells: role for short sequence homologies in the joining reaction.

“…The presence of short, 1-to 5-nucleotide homologies at roughly 60% of nonhomologous junctions has led several investigators to propose that these homologies play a role in nonhomologous recombination (1, 2,4,7,13,21,30,32,36). However, because these homologies are so short, it is difficult to distinguish between mechanistic relevance and chance occurrence.…”

A novel MVMp-based vector system specifically designed to reduce the risk of replication-competent virus generation by homologous recombination

“…9,11,24,25 Interestingly, nonhomologous recombination has also been implicated in the formation of the deletions known to arise spontaneously at a high rate in the MVMp genome. 26,27 Sequence analysis of the 5Ј and 3Ј recombination junctions should enable us to evaluate the relative contributions of the two recombination mechanisms in the production of contaminating helper genomes in our system. The 3Ј recombination junction is of particular interest, since a partially homologous (61%) 74-bp overlap remains in this region.…”

Nonhomologous recombination in the parvovirus chromosome: role for a CTATTTCT motif.

“…High-frequency deletion in the 5.0-kilobase linear singlestranded parvovirus chromosome proceeds via a nonhomologous recombination pathway that is mediated by 4-to 10-base-pair homologies (18). The deletions are nonrandom and range in size from approximately 2.0 to 4.5 kilobases (15,26).…”

“…Deletion variants are therefore readily obtained in encapsidated form and are separable from standard infectious virus by equilibrium density gradient centrifugation in CsCl (9,15). The DNA can be recovered from purified virus particles and cloned in plasmid (pBR322) or phage (M13) vectors, and the DNA sequences at the deletion junctions can be determined (18). Our analysis of three recombinants has shown that deletions averaging 3 kilobases in length occur between pairs of perfectly homologous 4-to 10-base-pair direct repeats such that one copy of the repeated sequence remains at the recombination junction (18).…”

“…The DNA can be recovered from purified virus particles and cloned in plasmid (pBR322) or phage (M13) vectors, and the DNA sequences at the deletion junctions can be determined (18). Our analysis of three recombinants has shown that deletions averaging 3 kilobases in length occur between pairs of perfectly homologous 4-to 10-base-pair direct repeats such that one copy of the repeated sequence remains at the recombination junction (18). In the present study we analyzed eight inde-pendently cloned recombination junctions obtained from naturally occurring deletion variants of the murine parvovirus minute virus of mice (MVM).…”