SOS factors involved in translesion synthesis - PubMed (original) (raw)

SOS factors involved in translesion synthesis

R L Napolitano et al. Proc Natl Acad Sci U S A. 1997.

Abstract

Mutations are permanent DNA sequence changes that can be induced when replication occurs on a damaged DNA template. In Escherichia coli, the process of translesion synthesis past a lesion that hinders replication requires the induction of SOS-controlled gene products, among which are those of the umuDC operon. To study translesion synthesis in vivo, we have constructed single-stranded vectors containing single 2-acetylaminofluorene adducts located within -1 and -2 frameshift mutation hot spots formed by short repetitive sequences. These adducts strongly hinder DNA replication as only 2-5% of the molecules give rise to progeny under non-SOS-induced conditions. Induction of the SOS response lead to a 10-fold increase in survival. Adducts present within repetitive sequences trigger the formation of misaligned primer/template replication intermediates which, upon elongation, will result in the fixation of frameshift errors (mutagenic translesion synthesis). Surprisingly we find that elongation from the nonslipped intermediate depends upon functional umuDC+ gene products, whereas elongation from the slipped intermediate is umuDC+ independent but requires another, as yet biochemically uncharacterized, SOS function. These data are discussed in terms of the different steps involved during translesion synthesis through a replication-blocking lesion.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Strategy for the construction of single-stranded DNA. Gapped-duplex intermediates were produced as previously described (20, 24). The gapped strand of the duplex was provided by a plasmid that was grown in a wild-type strain, whereas the complementary strand contained several uracil residues as the corresponding plasmid was propagated in a dut, ung strain (CJ 236) (25). Single AAF-adducted oligonucleotides were ligated into the gap, and the covalently closed circular plasmid was purified from CsCl equilibrium sedimentation. The uracil-containing strand was then selectively degraded using an enzymatic mixture containing uracil DNA glycosylase, and the 3′ → 5′ exonuclease of exonucleaseIII and T7 DNA polymerase (25). Constructions containing the G-AAF adduct (G*) within the sequence 5′-GGG*- or 5′-GGCG*CC- were made along with the corresponding control vectors (no adduct).

Figure 2

Figure 2

Structure of replication intermediates and involvement of SOS factors. (A) AAF-induced frameshift mutagenesis. When AAF adducts are situated in repetitive sequence contexts associated with both −1 and −2 frameshifts, the TLS intermediates can assume two forms: a nonslipped intermediate or a slipped intermediate. Elongation from the nonslipped intermediate, i.e., error-free elongation, proceeds from a lesion terminus at which the terminal nucleotide of the nascent strand is situated across from the AAF lesion (circled). Elongation from the lesion terminus appears to be stimulated by UmuDC. In contrast, frameshift mutations result from the elongation of the postlesion terminus of the slipped intermediate, at which the terminal nucleotide of the nascent strand is correctly paired with residues situated 5′ to the adduct on the template strand (shown in the square). G* is the AAF adducted guanine residue. (B) UV-induced base substitution mutagenesis. Both error-free and mutagenic TLS across a photoproduct involve elongation from a lesion terminus because, in the intermediate, the terminal nucleotide in the nascent strand is situated across from the lesion (circled). Both elongation events are strongly stimulated by the presence of UmuDC.

Similar articles

Cited by

References

    1. Livneh Z, Cohen-Fix O, Skaliter R, Elizur T. Crit Rev Biochem Mol Biol. 1993;28:465–513. - PubMed
    1. Higgins N P, Kato K, Strauss B. J Mol Biol. 1976;101:417–425. - PubMed
    1. Rupp W D, Wilde C E, III, Reno D L, Howard-Flanders P. J Mol Biol. 1971;61:25–44. - PubMed
    1. Koffel-Schwartz N, Coin F, Veaute X, Fuchs R P P. Proc Natl Acad Sci USA. 1996;93:7805–7810. - PMC - PubMed
    1. Belguise-Valladier P, Maki H, Sekiguchi M, Fuchs R P P. J Mol Biol. 1994;236:151–164. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources