Formation of heteroduplex DNA during mammalian intrachromosomal gene conversion.

Abstract

We have studied intrachromosomal gene conversion in mouse Ltk-cells with a substrate designed to provide genetic evidence for heteroduplex DNA. Our recombination substrate consists of two defective chicken thymidine kinase genes arranged so as to favor the selection of gene conversion products. The When uncorrected mispairs arise in duplex DNA, semiconservative DNA replication of the resultant heteroduplex segregates genetically distinct daughter molecules. Since one source for the generation of heteroduplex DNA is the process of genetic recombination, the two strands of a single DNA molecule can emerge from meiosis as nonidentical daughter molecules. Whereas four pairs of genes synapse during meiosis (4:4), fungal meiosis occasionally produces uneven aberrant segregations such as 5:3 or 3:5. Such postmeiotic segregations (PMS) are thought to result from the mitotic replication of heteroduplex DNA produced during genetic recombination.Most current models for genetic recombination derive from a model proposed by Holliday (15), who interpreted PMS as nonrepair of heteroduplex DNA (hDNA) and suggested that gene conversion resulted from correction of mismatches in hDNA. Although current models for meiotic recombination invoke different initiation mechanisms for gene conversion, such as single-strand invasion (22) or double-strand gap formation (30), these models generally suggest that heteroduplex formation may accompany gene conversion. In fungi, the most compelling genetic evidence for hDNA is the observation of PMS during meiotic recombination (16, 26) or of sectored colonies during mitotic recombination (8,11,28,36). Such recombinants most likely result from the failure to repair hDNA intermediates prior to DNA replication.Evidence for the existence of heteroduplex DNA in mammalian cells is primarily indirect. Results of both in vivo and in vitro studies indicate that mammalian cells do possess the ability to process preformed mismatched DNA, hDNA, to the homoduplex forms following transfection (1,2,6,9,10,13,32,33

Increase of spontaneous intrachromosomal homologous recombination in mammalian cells expressing a mutant p53 protein

“…Heteroduplex DNA is a common intermediate molecule predicted by all the homologous recombination models (Holliday, 1964;Meselson and Radding, 1975;Szostak et al, 1983). In mammalian cells, heteroduplex DNA as a recombination intermediate has been described in in vitro reactions with human nuclear extracts (Lopez et al, 1987) as well as in cultured mouse L cells lines, similar to the pJS3-10 line used here (Bollag et al, 1992). In the present experiments, recombination between the two TK sequences should produce a heteroduplex DNA intermediate bearing a 8 bp long loop.…”

Testing Predictions of the Double-Strand Break Repair Model Relating to Crossing Over in Mammalian Cells

“…Within the vector-borne C region of homology, ferred mode involving the cutting of like strands in one of which there is newly synthesized DNA near the there are three unique restriction enzyme sites for introducing the recombination-initiating DSB, and at regular junction [ Figure 1F(i); Gilbertson and Stahl 1996;Foss et al 1999;Baker and Birmingham 2001]. In yeast intervals the vector-borne C region is marked by small palindrome insertions, which are semirefractory to MMR meiosis, Holliday junction cleavage in the preferred sense is observed in ‫%98-48ف‬ of crossover events (Foss et al (Nag et al 1989;Bollag et al 1992;Donoho et al 1998;Li and Baker 2000). The palindrome insertions facilitated 1999; Merker et al 2003).…”

Analysis of One-Sided Marker Segregation Patterns Resulting From Mammalian Gene Targeting

“…However, what repair system acts to remove palindrome mismatches when the threshold distance from the nick to the palindrome is exceeded? Two mechanisms are possible: (i) junction cuts (or, possibly, unligated 39-ends; Stahl et al 2004) at positions 29 and 49 direct removal of DSB proximal palindromes (early repair of hDNA) (McGill et al 1989;Haber et al 1993;Foss et al 1999) and (ii) palindromes are removed by localized (nick-independent) repair (Weiss and Wilson 1987;Bollag et al 1992;Bill et al 2001). With regard to mechanism (i), nicks at positions 29 and 49 would be located at variable distances from the palindrome mismatches due to the different lengths of homology in the four gene-targeting vectors used in generating the recombinants.…”

“…In each vector, the Cm region of homology contains two NotI palindrome markers that replace endogenous BamHI and ApaI sites located 598 and 439 bp, respectively, from the unique BstXI site of vector linearization. Since small palindromes are semirefractory to mismatch repair in yeast (Nag et al 1989) and mammalian cells (Bollag et al 1992;Donoho et al 1998;Li and Baker 2000), their segregation patterns provide information about hDNA formation in recombination intermediates. The vectors differ in the amount of homology that they share with the chromosomal Ig Cm region, although in each it is well above the 1-kb minimum required to effect mammalian gene targeting (Shulman et al 1990;Hasty et al 1991).…”

“…Consequently, different segregation classes are possible in crosses that involve markers flanking a DSB. To reveal heteroduplexes, 1 genetic crosses often utilize small palindromes as markers, since they are semirefractory to repair when in heteroduplex with the wild-type sequence (Nag et al 1989;Bollag et al 1992;Donoho et al 1998;Li and Baker 2000). In describing marker segregation patterns that arise during gene targeting, we have found it convenient (Birmingham et al 2004) to adopt nomenclature normally used to categorize meiotic recombination events in yeast and fungi (Petes et al 1991).…”