Biopolym. Cell. 2012; 28(3):218-222.
http://dx.doi.org/10.7124/bc.000051
Structure and Function of Biopolymers
Study of the activity of DNA polymerases β and λ using 5-formyluridine containing DNA substrates
1Belousova E. A., 1Lavrik O. I.
  1. Novosibirsk Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences
    8, Akademika Lavrentieva Ave., Novosibirsk, Russian Federation, 630090

Abstract

Aims. To investigate the TLS-activity of human DNA polymerases β and λ (pols β and λ) using 5-formyluridine (5-foU) containing DNA duplexes which are imitating the intermediates during replication of the leading DNA strand, and to study the influence ofreplication factors hRPA and hPCNA on this activity. Methods. The EMSA and the methods of enzyme’s kinetics were used. Results. The capability of pols β and λ to catalyze DNA synthesis across 5-foU was investigated and the kinetic characteristics of this process in the presence and in the absence of protein factors hRPA and hPCNA were evaluated. Conclusions. It was shown that: (i) both proteins are able to catalyze TLS on used DNA substrates regardless of the reaction conditions, however, pol λ was more accurate enzyme; (ii) hRPA can stimulate the efficacy of the nonmutagenic TLS catalyzed by pol λ at the nucleotide incorporation directly opposite of 5-foU, at the same time it doesn’t influence the incorporation efficacy if the damage displaced into the duplex; (iii) hPCNA doesn’t influence the efficacy of TLS catalyzed by both enzymes.
Keywords: translesion synthesis, DNA polymerases β and λ, 5-formyluridine
Full text: (PDF, in English)

References

[1] Bjelland S., Anensen H., Knaevelsrud I., Seeberg E. Cellular effects of 5-formyluracil in DNA Mutat. Res 2001 486, N 2:147–154.
[2] Ohmori H., Friedberg E. C., Fuchs R. P., Goodman M. F., Hanaoka F., Hinkle D., Kunkel T. A., Lawrence C. W., Livneh Z., Nohmi T., Prakash L., Prakash S., Todo T., Walker G. C., Wang Z., Woodgate R. The Y-family of DNA polymerases Mol. Cell 2001 8, N 1:7–8.
[3] Belousova E. A., Lavrik O. I. DNA polymerases beta and lambda, and their roles in the DNA replication and repair Mol. Biol. (Moscow) 2010 44, N 6:947–965.
[4] Maga G., Villani G., Crespan E., Wimmer U., Ferrari E., Bertocci B., Hubscher U. 8-oxo-guanine bypass by human DNA polymerases in the presence of auxiliary proteins Nature 2007 447, N 7144:606–608.
[5] Braithwaite E. K., Prasad R., Shock D. D., Hou E. W., Beard W. A., Wilson S. H. DNA polymerase lambda mediates a back-up base excision repair activity in extracts of mouse embryonic fibroblasts J. Biol. Chem 2005 280, N 18:18469–18475.
[6] Drachkova I. A., Petruseva I. O., Safronov I. V., Zakharenko A. L., Shishkin G. V., Lavrik O. I., Khodyreva S. N. Reagents for modification of protein-nucleic acids complexes. II. Site-specific photomodification of DNA-polymerase beta complexes with primers elongated by the dCTP exo-N-substituted arylazido derivatives Bioorg. Khim 2001 27, N 3:197–204.
[7] Mazin A. Label's introducing into DNA Methods of molecular genetics and gene engineering] / Ed. R. I. Salganik Novosibirsk: Science SBRAS, 1990:25–26.
[8] Belousova E. A., Maga G., Fan Y., Kubareva E. A., Romanova E. A., Lebedeva N. A., Oretskaya T. S., Lavrik O. I. DNA polymerases beta and lambda bypass thymine glycol in gapped DNA structures Biochemistry 2010 49, N 22:4695–4704.
[9] Kraev A. Sequencing gels Methods of molecular genetics and gene engineering / Ed. R. I. Salganik Novosibirsk: Science SBRAS, 1990:145–153.
[10] Shtygasheva A. A., Belousova E. A., Rechkunova N. I., Lebedeva N. A., Lavrik O. I. DNA polymerases beta and lambda as potential participants of TLS during genomic DNA replication on the lagging strand Biochemistry (Moscow) 2008 73, N 11 P. 1207–1213.
[11] Blanca G., Villani G., Shevelev I., Ramadan K., Spadari S., Hubscher U., Maga G. Human DNA polymerases lambda and beta show different efficiencies of translesion DNA synthesis past abasic sites and alternative mechanisms for frameshift generation Biochemistry 2004 43, N 36:11605–11615.
[12] Kunkel T. A., Alexander P. S. The base substitution fidelity of eukaryotic DNA polymerases. Mispairing frequencies, site preferences, insertion preferences, and base substitution by dislocation J. Biol. Chem 1986 261, N 1:160–166.
[13] Fanning E., Klimovich V., Nager A. R. A dynamic model for replication protein A (RPA) function in DNA processing pathways Nucleic Acids Res 2006 34, N 15:4126–4137.
[14] Maga G., Hubscher U. Proliferating cell nuclear antigen (PCNA): a dancer with many partners J. Cell Sci 2003 116, Pt 15 P. 3051–3060.
[15] Budzowska M., Kanaar R. Mechanisms of dealing with DNA damage-induced replication problems Cell Biochem. Biophys 2009 53, N 1:17–31.
[16] Kedar P. S., Kim S. J., Robertson A., Hou E., Prasad R., Horton J. K., Wilson S. H. Direct interaction between mammalian DNA polymerase beta and proliferating cell nuclear antigen J. Biol. Chem 2002 277, N 34:31115–31123.
[17] Shimazaki N., Yazaki T., Kubota T., Sato A., Nakamura A., Kurei S., Toji S., Tamai K., Koiwai O. DNA polymerase lambda directly binds to proliferating cell nuclear antigen through its confined C-terminal region Genes Cells 2005 10, N 7:705– 715.
[18] Maga G., Villani G., Ramadan K., Shevelev I., Tanguy Le Gac N., Blanco L., Blanca G., Spadari S., Hubscher U. Human DNA polymerase lambda functionally and physically interacts with proliferating cell nuclear antigen in normal and translesion DNA synthesis J. Biol. Chem 2002 277, N 50:48434–48440.
[19] Maga G., Shevelev I., Villani G., Spadari S., Hubscher U. Human replication protein A can suppress the intrinsic in vitro mutator phenotype of human DNA polymerase lambda Nucleic Acids Res 2006 34, N 5:1405–1415.
[20] Crespan E., Hubscher U., Maga G. Error-free bypass of 2-hydroxyadenine by human DNA polymerase lambda with Proliferating Cell Nuclear Antigen and Replication Protein A in different sequence contexts Nucleic Acids Res 2007 35, N 15 P. 5173–5181.
[21] Krasikova Y. S., Belousova E. A., Lebedeva N. A., Pestryakov P. E., Lavrik O. I. Interaction between DNA Polymerase lambda and RPA during translesion synthesis Biochemistry (Moscow) 2008 73, N 9:1042–1046.