Saving the Ends for Last: The Role of Pol μ in DNA End Joining
2005; Elsevier BV; Volume: 19; Issue: 3 Linguagem: Inglês
10.1016/j.molcel.2005.07.008
ISSN1097-4164
Autores Tópico(s)Carcinogens and Genotoxicity Assessment
ResumoAt least three DNA polymerases participate in nonhomologous end joining in mammalian cells: pol μ, pol κ, and TdT. A study in this issue of Molecular Cell (Nick McElhinny et al., 2005Nick McElhinny, S.A., Havener, J.M., Garcia-Diaz, M., Juarez, R., Bebenek, K., Kee, B.L., Blanco, L., Kunkel, T.A., and Ramsden, D.A. (2005). Mol. Cell 19, this issue, 357–366.Google Scholar) clarifies the role of pol μ in end joining at the kappa light chain locus and also provides a biochemical explanation for the unique polymerization functions of pol μ on DNA ends. At least three DNA polymerases participate in nonhomologous end joining in mammalian cells: pol μ, pol κ, and TdT. A study in this issue of Molecular Cell (Nick McElhinny et al., 2005Nick McElhinny, S.A., Havener, J.M., Garcia-Diaz, M., Juarez, R., Bebenek, K., Kee, B.L., Blanco, L., Kunkel, T.A., and Ramsden, D.A. (2005). Mol. Cell 19, this issue, 357–366.Google Scholar) clarifies the role of pol μ in end joining at the kappa light chain locus and also provides a biochemical explanation for the unique polymerization functions of pol μ on DNA ends. Repair of broken DNA molecules by ligation, a process known as nonhomologous end-joining (NHEJ), constitutes a major pathway of DNA double-strand break (DSB) repair in mammalian cells and is essential for the elaborate cascade of chromosomal breakage and rejoining events that occur during immunoglobulin gene rearrangement. The structures of DNA ends produced in vivo, however, are rarely amenable to a simple ligation and often have single-stranded overhangs and may contain damaged bases or sugar moieties that require processing. The assembly of proteins that mediates NHEJ must therefore be extremely creative and flexible, able to accommodate different structures, and mediate ligation without incurring excessive deletion of adjacent sequences. In this issue of Molecular Cell, Ramsden and colleagues present novel insights into the DNA processing events that require DNA polymerization across a DSB and suggest that the versatility of these DNA polymerases may derive from their ability to "see" the template strand very differently from a replicative DNA polymerase (Nick McElhinny et al., 2005Nick McElhinny, S.A., Havener, J.M., Garcia-Diaz, M., Juarez, R., Bebenek, K., Kee, B.L., Blanco, L., Kunkel, T.A., and Ramsden, D.A. (2005). Mol. Cell 19, this issue, 357–366.Google Scholar). Nick McElhinny et al., 2005Nick McElhinny, S.A., Havener, J.M., Garcia-Diaz, M., Juarez, R., Bebenek, K., Kee, B.L., Blanco, L., Kunkel, T.A., and Ramsden, D.A. (2005). Mol. Cell 19, this issue, 357–366.Google Scholar compared the activities of pol μ and pol λ, two members of the Pol X family of small, nonreplicative DNA polymerases, and found that only pol μ could polymerize across a DSB with no pairing between the primer and the template (see Figure 1C ). In contrast, both pol μ and pol λ could perform gap-filling synthesis using mismatched ends aligned with a 2 bp overlap. Using a pre-B cell line induced to undergo immunoglobulin gene rearrangement, the authors also found that overexpression of pol μ, but not pol λ, substantially reduced the number of nucleotides deleted from coding junctions during Vκ-Jκ joining. These findings correlate well with previous observations that pol μ−/− mice exhibit a specific deficiency in B cell differentiation caused by excessive deletion at Vκ-Jκ and Vλ-Jλ coding junctions (Bertocci et al., 2003Bertocci B. De Smet A. Berek C. Weill J.C. Reynaud C.A. Immunity. 2003; 19: 203-211Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Pol λ−/− mice, on the other hand, do not exhibit any significant deficiencies in immune function (Bertocci et al., 2002Bertocci B. De Smet A. Flatter E. Dahan A. Bories J.C. Landreau C. Weill J.C. Reynaud C.A. J. Immunol. 2002; 168: 3702-3706PubMed Google Scholar, Kobayashi et al., 2002Kobayashi Y. Watanabe M. Okada Y. Sawa H. Takai H. Nakanishi M. Kawase Y. Suzuki H. Nagashima K. Ikeda K. Motoyama N. Mol. Cell. Biol. 2002; 22: 2769-2776Crossref PubMed Scopus (117) Google Scholar). The data from Nick McElhinny et al. show that the catalytic activity of pol μ is essential for the suppression of coding end deletions, suggesting that use of the 3′ ends as primers actively prevents deletions by preparing the ends for ligation. The ability of pol μ to add nucleotides without a paired template is arguably distinct from the untemplated DNA polymerization carried out by terminal deoxytransferase (TdT), another Pol X family member most closely related to pol μ. TdT is exclusively expressed in cells of the developing immune system and plays a critical role in diversification of immunoglobulin genes by adding untemplated bases to 3′ strands of coding ends. These random "N" additions occur only at V(D)J junctions in immunoglobulin and T cell receptor genes and are not seen in TdT−/− lymphocytes (Benedict et al., 2000Benedict C.L. Gilfillan S. Thai T.H. Kearney J.F. Immunol. Rev. 2000; 175: 150-157Crossref PubMed Google Scholar). Nick McElhinny et al. demonstrate that the additions made by pol μ in vitro are not random N additions but are primarily template dependent, the template in this case being the 3′ strand of another DNA end (see Figure 1C). TdT levels are high during immunoglobulin heavy chain joining but low during light chain rearrangement, suggesting that TdT and pol μ may act sequentially during V(D)J recombination. The use of pol μ during light chain processing may reflect the need to restrain deletions of the small light chain gene and to prevent extensive insertions. The differences in template requirements of pol μ, pol λ, and TdT may stem from loop 1, a single small domain that is present in TdT and pol μ, shorter in pol λ, and completely absent in the template-dependent polymerase pol β. In the crystal structure of murine TdT, loop 1 forms a lariat that extrudes from the polymerase at a position normally occupied by the template strand in a template-directed polymerase (Delarue et al., 2002Delarue M. Boule J.B. Lescar J. Expert-Bezancon N. Jourdan N. Sukumar N. Rougeon F. Papanicolaou C. EMBO J. 2002; 21: 427-439Crossref PubMed Scopus (112) Google Scholar). Nick McElhinny et al. show that deletion of loop 1 from pol μ abrogates its ability to perform synthesis from unpaired primer-template combinations yet does not affect synthesis from partially aligned ends. A pol μ mutant lacking loop 1 also fails to suppress deletions during Vκ-Jκ joining in cells, similar to wild-type pol λ, further suggesting that the pairing-independent activities of pol μ are most essential in vivo. The sequence of loop 1 in TdT is very different from pol μ, however, such that the loop in pol μ is unlikely to form a similar conformation as the loop in TdT. From the current data, one would expect that pol μ loop 1 would accommodate a single-stranded overhang from the opposite DNA end, but this question will only be resolved with structure determination of pol μ. The study by Ramsden and colleagues answers several important questions about the role of pol μ in end joining, but there are many issues still left to be resolved, particularly with respect to the distribution of labor between pol μ and pol λ. Both polymerases are widely expressed in mammalian tissues, yet neither pol μ−/− nor pol λ−/− cells are sensitive to DSB-inducing agents, which would seem to contradict the idea that they are each responsible for specific aspects of DNA processing during NHEJ. It is possible that pol μ and pol λ may share a set of redundant roles in general aspects of NHEJ processing or that polymerization during NHEJ is not as essential as the other catalytic activities. A previous study using cell extracts showed that immunodepletion of pol λ, but not of pol μ, abolished joining of mismatched DNA ends in vitro (Lee et al., 2004Lee J.W. Blanco L. Zhou T. Garcia-Diaz M. Bebenek K. Kunkel T.A. Wang Z. Povirk L.F. J. Biol. Chem. 2004; 279: 805-811Crossref PubMed Scopus (177) Google Scholar). In contrast, Lieber and colleagues found that either pol μ or pol λ could mediate mismatched end joining in a purified system, with pol μ playing a more dominant role when both polymerases were present (Ma et al., 2004Ma Y. Lu H. Tippin B. Goodman M.F. Shimazaki N. Koiwai O. Hsieh C.L. Schwarz K. Lieber M.R. Mol. Cell. 2004; 16: 701-713Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). Although further investigation is clearly required to resolve these issues, it may be the case that the contribution of each polymerase depends on the sequence context and overhang structure of the DNA ends. Data from Wilson and colleagues support this view, as pol μ and pol λ showed variable abilities to complement end joining of mismatched 3′ overhangs in yeast strains deficient in Pol X family member Pol IV, depending on the length and position of homology available in the overhangs (Daley et al., 2005Daley, J.M., Vander Laan, R.L., Suresh, A., and Wilson, T.E. (2005). J. Biol. Chem., in press. Published online June 17, 2005. 10.1074/jbc.M505277200.Google Scholar). Yet another possibility is suggested by a recent study demonstrating that pol λ−/− cells are hypersensitive to oxidative damage and colocalize with components of the excision repair pathway (Braithwaite et al., 2005Braithwaite, E.K., Kedar, P.S., Lan, L., Polosina, Y.Y., Asagoshi, K., Poltoratsky, V.P., Horton, J.K., Miller, H., Teebor, G.W., Yasui, A., and Wilson, S.H. (2005). J. Biol. Chem., in press. Published online July 7, 2005. 10.1074/jbc.C500256200.Google Scholar), which could mean that multiple repair pathways are dependent on these unusual polymerases.
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