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

Abstract:

:Although DNA breakage and reunion in nonhomologous recombination are poorly understood, previous work suggests that short sequence homologies may play a role in the end-joining step in mammalian cells. To study the mechanism of end joining in more detail, we inserted a polylinker into the simian virus 40 T-antigen intron, cleaved the polylinker with different pairs of restriction enzymes, and transfected the resulting linear molecules into monkey cells. Analysis of 199 independent junctional sequences from seven constructs with different mismatched ends indicates that single-stranded extensions are relatively stable in monkey cells and that the terminal few nucleotides are critical for cell-mediated end joining. Furthermore, these studies define three mechanisms for end joining: single-strand, template-directed, and postrepair ligations. The latter two mechanisms depend on homologous pairing of one to six complementary bases to position the junction. All three mechanisms operate with similar overall efficiencies. The relevance of this work to targeted integration in mammalian cells is discussed.

journal_name

Mol Cell Biol

authors

Roth DB,Wilson JH

doi

10.1128/mcb.6.12.4295

subject

Has Abstract

pub_date

1986-12-01 00:00:00

pages

4295-304

issue

12

eissn

0270-7306

issn

1098-5549

journal_volume

6

pub_type

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