(NAS Colloquium) Links Between Recombination and Replication: Vital Roles of Recombination

The following HTML text is provided to enhance online readability. Many aspects of typography translate only awkwardly to HTML. Please use the page image as the authoritative form to ensure accuracy.

Colloquium on Links Between Recombination and Replication: Vital Roles of Recombination

Colloquium

Accuracy of lesion bypass by yeast and human DNA polymerase η

M.Todd Washington, Robert E.Johnson, Louise Prakash, and Satya Prakash*

Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, TX 77555–1061

DNA polymerase η (Polη) functions in the error-free bypass of UV-induced DNA lesions, and a defect in Polη in humans causes the cancer-prone syndrome, the variant form of xeroderma pigmentosum. Both yeast and human Polη replicate through a cis-syn thymine-thymine dimer (TT dimer) by inserting two As opposite the two Ts of the dimer. Polη, however, is a low-fidelity enzyme, and it misinserts nucleotides with a frequency of ≈10⁻² to 10⁻³ opposite the two Ts of the TT dimer as well as opposite the undamaged template bases. This low fidelity of nucleotide insertion seems to conflict with the role of Polη in the error-free bypass of UV lesions. To resolve this issue, we have examined the ability of human and yeast Polη to extend from paired and mispaired primer termini opposite a TT dimer by using steady-state kinetic assays. We find that Polη extends from mispaired primer termini on damaged and undamaged DNAs with a frequency of ≈10⁻² to 10⁻³ relative to paired primer termini. Thus, after the incorporation of an incorrect nucleotide, Polη would dissociate from the DNA rather than extend from the mispair. The resulting primer-terminal mispair then could be subject to proofreading by a 3′→5′ exonuclease. Replication through a TT dimer by Polη then would be more accurate than that predicted from the fidelity of nucleotide incorporation alone.

The presence of a DNA lesion in the template strand blocks the normal replication machinery. Such lesions can be bypassed by the action of specialized translesion synthesis DNA polymerases (Pols) (1), or by a “copy choice” type of DNA synthesis in which the newly synthesized daughter strand of the undamaged complementary sequence is used as a template to bypass the lesions (2). Alternatively, recombinational mechanisms may be used (3).

The RAD6-Dependent Pathways of Damage Bypass

As indicated from genetic studies in the yeast Saccharomyces cerevisiae, the RAD6 and RAD18 genes are indispensable for error-free as well as mutagenic bypass processes in eukaryotes (4, 5). Rad6, a ubiquitin-conjugating enzyme, forms a tight complex with Rad18, a DNA-binding protein (6, 7). Although the role of the Rad6–Rad18 complex in damage bypass is not known, one possibility is that it modulates the turnover of the replicative Pol stalled at the lesion site and thereby promotes the entry of translesion synthesis Pols to the lesion site. The Rad6–Rad18-dependent bypass of UV lesions involves at least three separate branches, wherein the RAD5 and RAD30 genes function in alternate error-free bypass pathways and REV3 functions in mutagenic bypass (8–10). Rad5, a DNA-dependent ATPase (11), is a member of the Swi-Snf family of proteins (8), but the manner of its action in damage bypass is unknown. RAD30, a member of the umuC/dinB family (9, 12), encodes a DNA Pol, Polη (13), which has the unique ability to replicate through a diversity of DNA lesions.

The Rev3 protein, together with Rev7, constitutes DNA Polζ (14). Polζ also functions in translesion synthesis, but its role in lesion bypass is quite specific. The indispensability of the REV3 and REV7 genes for mutagenesis induced by UV light and other DNA-damaging agents (15, 16) had prompted the generally held notion that Polζ would be a very low-fidelity Pol capable of bypassing DNA lesions. Steady-state kinetic studies, however, have shown that Polζ has a fairly high fidelity, as it misincorporates nucleotides opposite undamaged template bases with a frequency of ≈10⁻⁴ to 10⁻⁵ (17). The fidelity of nucleotide incorporation of Polζ is about the same as that of DNA Polα, required for lagging strand DNA synthesis. Polζ is very inefficient at inserting nucleotides opposite the 3′T of the thymine-thymine dimer (TT dimer) or the (6–4) TT photoproduct, and it is also very poor at inserting nucleotides opposite abasic sites; consequently, Polζ bypasses these lesions very inefficiently (17). Polζ, however, is a very efficient extender of base mispairs, and its ability to extend from base mispairs is about 1,000-fold better than its ability to insert a mispaired base (f_inc ≈10⁻⁴ to 10⁻⁵) (17). Importantly, Polζ is also very adept at extending from nucleotides placed opposite DNA lesions. For example, Polζ extends from a G placed opposite the 3′T of a TT dimer or a (6–4) TT photoproduct almost as efficiently as it extends from an A placed opposite an undamaged T (17). These and other observations have led to the formulation of the principle that mutagenic bypass in eukaryotes involves the action of two different DNA Pols in which a DNA Pol, as for example, human RAD30B-encoded Polι (1), inserts a nucleotide opposite the DNA lesion, whereas Polζ subsequently extends from the inserted nucleotide (17). The indispensability of Polζ for mutagenic bypass in S. cerevisiae derives from the fact that this is the sole enzyme responsible for the extension of “wrong” nucleotides placed opposite DNA lesions.

Role of DNA Polη in the Error-Free Bypass of UV Lesions

Genetic studies in S. cerevisiae have indicated a role for RAD30-encoded Polη in the error-free bypass of UV lesions. Although the rad30∆ mutation confers a moderate degree of UV sensitivity, a synergistic increase in UV sensitivity occurs in the rad5∆ rad30∆ double mutant, and the frequency of UV-induced mutations is much higher in the double mutant than in the rad5∆ or rad30∆ single mutants (9, 10). UV light induces the formation of cyclobutane TT dimers, and Polη efficiently replicates through the TT dimer by inserting two As opposite the two Ts of the dimer (13). UV, however, also induces the formation of lesions at 5′-TC-3′ and 5′-CC-3′ dipyrimidine sites, and the 3′C in both these sequence contexts is highly mutagenic. In both yeast and humans, UV-induced mutations occur predominantly by a 3′ C → T transition that results from the insertion of an A opposite the 3′C during DNA replication (18). In vitro bypass studies with a TC or CC cis-syn

This paper results from the National Academy of Sciences colloquium, “Links Between Recombination and Replication: Vital Roles of Recombination,” held November 10–12, 2000, in Irvine, CA.

Abbreviations: Pol, polymerase; TT dimer, thymine-thymine dimer; 8-oxoG, 7,8-dihydro 8-oxoguanine; m6G, ⁶O-methylguanine.

*	To whom reprint requests should be addressed at: Sealy Center for Molecular Science, University of Texas Medical Branch, 6.104 Medical Research Building, 11th and Mechanic Streets, Galveston, TX 77555–1061. E-mail: sprakash@scms.utmb.edu.

Free Resources

Related Titles