Lesion Bypass Activities of Human DNA Polymerase μ
Journal of Biological Chemistry
DNA polymerase (Pol) is a newly discovered member of the polymerase X family with unknown cellular function. The understanding of Pol function should be facilitated by an understanding of its biochemical activities. By using purified human Pol for biochemical analyses, we discovered the lesion bypass activities of this polymerase in response to several types of DNA damage. When it encountered a template 8-oxoguanine, abasic site, or 1,N 6 -ethenoadenine, purified human Pol efficiently bypassed
... he lesion. Even bulky DNA adducts such as N-2-acetylaminofluorene-adducted guanine, (؉)and (؊)-trans-anti-benzo[a]pyrene-N 2 -dG were unable to block the polymerase activity of human Pol. Bypass of these simple base damage and bulky adducts was predominantly achieved by human Pol through a deletion mechanism. The Pol specificity of nucleotide incorporation indicates that the deletion resulted from primer realignment before translesion synthesis. Purified human Pol also effectively bypassed a template cis-syn TT dimer. However, this bypass was achieved in a mainly error-free manner with AA incorporation opposite the TT dimer. These results provide new insights into the biochemistry of human Pol and show that efficient translesion synthesis activity is not strictly confined to the Y family polymerases. DNA polymerase (Pol) 1 is a newly discovered member of the X family polymerases (1, 2). Additional members in this family include Pol␤, Pol, and terminal deoxynucleotidyltransferase (1-3). During base excision repair in higher eukaryotes, Pol␤ is a major repair synthesis polymerase (4 -6). Terminal deoxynucleotidyltransferase catalyzes nucleotide additions to DNA in a template-independent manner (7, 8). This enzyme functions during V(D)J recombination of the immunoglobulin genes and T-cell receptor genes and is restricted to lymphoid tissues (7-9). Cellular functions of Pol and Pol have not been clearly defined. Although the biochemical activities of the X family DNA polymerases appear to be quite diverse, all of the Y family DNA polymerases share a common biochemical activity: synthesis opposite DNA lesions (reviewed in Refs. 10 -13). In eukaryotes, the Y family consists of REV1 and DNA polymerases , , and (14) . Thus, it is generally believed that a major function of the Y family DNA polymerases is to copy damaged sites of DNA during replication, a cellular process referred to as lesion bypass or translesion synthesis. Genetic studies indicate that REV1 (15-18) and Pol (19 -22) are indeed involved in lesion bypass in cells. Lesion bypass can be error-free as a result of insertion of the correct nucleotide opposite the lesion or errorprone as the result of insertion of an incorrect nucleotide opposite the lesion. Both error-free and error-prone nucleotide insertions have been observed with the Y family polymerases depending on the specific lesion and the specific polymerase (reviewed in Refs. 10 -12). Biochemical studies of purified human Pol have uncovered a unique property that has never been observed with any other polymerases studied so far (23). Human Pol is highly prone to frameshift DNA synthesis (23). At single-nucleotide repeat sequences, DNA synthesis by human Pol is mediated mainly by a deletion mechanism because of primer-template realignment before synthesis (23). Furthermore, when the primer 3Ј end contains one or a few mismatches, human Pol can promote primer-template realignment such that the primer 3Ј end can find its complementary sequences on the template several nucleotides downstream, achieving microhomology search and microhomology pairing (23). These striking biochemical properties led Zhang et al. (23) to propose that Pol may be involved in nonhomologous end joining (NHEJ) for double-strand DNA repair. The biochemical properties of human Pol ruled out a significant role for this polymerase in somatic hypermutation during immunoglobulin development. One important cause of DNA double-strand breaks is DNA damage. It is conceivable that some damaged sites may contain clustered lesions or that base damage may be contained near some double-strand DNA breaks. Under those circumstances, Pol would encounter DNA base damage while performing microhomology search and pairing, as well as DNA synthesis, during NHEJ. Hence, we asked whether Pol is capable of translesion synthesis. In this report, we demonstrate that human Pol indeed possesses efficient lesion bypass activities in response to very different types of DNA damage, ranging from simple base modifications and baseless sites to bulky chemical DNA adducts and cis-syn TT dimer of UV radiation. Although in vitro bypass of a template TT dimer is achieved by human Pol in an error-free manner, bypass of the other tested lesions is mediated by a deletion mechanism that effectively avoids copying the damaged template base through primer realignment. These findings provide new insights into the biochemistry of human Pol and show that efficient translesion synthesis activity is not strictly confined to Y family polymerases.