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The multifaceted roles of family X DNA polymerases

 

 

In the last years, the number of identified eukaryotic DNA polymerases (pols) increased from the so called “classical” five pols α, β, γ, δ and ε to more than fifteen. Based on sequence homology, some of these “novel” pols have been grouped into a completely new family, called family Y, comprising pol η, ι, κ and Rev1. In addition, new members of the previously recognised families B (pols ζ), X (pols λ, μ) and A (pol θ, pol ν) have been identified. A clear understanding of their physiological role(s) is hampered by the substantial lack of knowledge of their biochemical properties, mainly due to the fact that most of these pols have been identified at the genetic level, but the products of the respective genes have never been purified from cells.

We have focused our attention on the DNA polymerase family X. A schematic diagram of the conserved domains/functions among the members of this family is shown below

 





 

 

We have focused on the Pol λ enzyme. Our studies have shown the following properties:

 








 

We have found that Pol λ is important for a specialized translesion synthesis across the oxidative lesion 8-oxo-G, during the MutYH-dependent post-replicational repair of A:8-oxo-G mismatches generated during DNA replication








 

Our recent results show that Pol λ is the most faithful in bypassing an 8-oxo-G lesion, suggesting a role in the MYH-dependent BER of A:8-oxo-G mismatches as depicted below.





 

We have also found that Pol λ has the ability to promote the annealing and elongation of DNA strands bearing terminal microhomologies (2-5 nt). This suggests a possible role of Pol λ in the alternative double strand break repair mechanism called Microhomology-Mediated End Joining (MMEJ)





 

However, on DNA strands with the (CAG)n repeat, which is found in the pathological version of the Huntingtin gene, mutated in the neurological disorder Huntington disease, Pol β, another family X member, was the most proficient, leading to expansion of the triplet repeats. These results suggested that Pol β may be responsible for CAG somatic expansion during MMEJ repair of DNA breaks in neurons.





 

We have also found that Pol λ is important in protecting human cells against replicative stress and it is functionally linked to the intra S-phase checkpoint mediated by ATR and Chk1

 





 

References:

 

Zucca E, Bertoletti F, Wimmer U, Ferrari E, Mazzini G, Khoronenkova S, Grosse  N, van Loon B, Dianov G, Hübscher U, Maga G. Silencing of human DNA polymerase λ  causes replication stress and is synthetically lethal with an impaired S phase checkpoint. Nucleic Acids Res. 2013 Jan 7;41(1):229-41.

 

Crespan E, Pasi E, Imoto S, Hübscher U, Greenberg MM, Maga G. Human DNA polymerase β, but not λ, can bypass a 2-deoxyribonolactone lesion together with proliferating cell nuclear antigen. ACS Chem Biol. 2013 Feb 15;8(2):336-44.

 

Crespan E, Czabany T, Maga G, Hübscher U. Microhomology-mediated DNA strand annealing and elongation by human DNA polymerases λ and β on normal and repetitive DNA sequences. Nucleic Acids Res. 2012 Jul;40(12):5577-90.

 

Amoroso A, Concia L, Maggio C, Raynaud C, Bergounioux C, Crespan E, Cella R, Maga G. Oxidative DNA damage bypass in Arabidopsis thaliana requires DNA polymerase λ and proliferating cell nuclear antigen 2. Plant Cell. 2011 Feb;23(2):806-22. doi: 10.1105/tpc.110.081455.

 

Maga G, Crespan E, Wimmer U, van Loon B, Amoroso A, Mondello C, Belgiovine C,  Ferrari E, Locatelli G, Villani G, Hübscher U. Replication protein A and proliferating cell nuclear antigen coordinate DNA polymerase selection in 8-oxo-guanine repair. Proc Natl Acad Sci U S A. 2008 Dec 30;105(52):20689-94. 

 

Crespan E, Hübscher 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(15):5173-81.

 

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 May 31;447(7144):606-8.

 

Crespan E, Alexandrova L, Khandazhinskaya A, Jasko M, Kukhanova M, Villani G,  Hubscher U, Spadari S, Maga G. Expanding the repertoire of DNA polymerase substrates: template-instructed incorporation of non-nucleoside triphosphate analogues by DNA polymerases beta and lambda. Nucleic Acids Res. 2007;35(1):45-57.

 

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 Mar 6;34(5):1405-15.

 

Crespan E, Zanoli S, Khandazhinskaya A, Shevelev I, Jasko M, Alexandrova L, Kukhanova M, Blanca G, Villani G, Hubscher U, Spadari S, Maga G. Incorporation of non-nucleoside triphosphate analogues opposite to an abasic site by human DNA polymerases beta and lambda. Nucleic Acids Res. 2005 Jul 25;33(13):4117-27.

 

Maga G, Ramadan K, Locatelli GA, Shevelev I, Spadari S, Hubscher U. DNA elongation by the human DNA polymerase lambda polymerase and terminal transferase activities are differentially coordinated by proliferating cell nuclear antigen and replication protein A. J Biol Chem. 2005 Jan 21;280(3):1971-81. 

 

Maga G, Blanca G, Shevelev I, Frouin I, Ramadan K, Spadari S, Villani G, Hubscher U. The human DNA polymerase lambda interacts with PCNA through a domain important for DNA primer binding and the interaction is inhibited by p21/WAF1/CIP1. FASEB J. 2004 Nov;18(14):1743-5.

 

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 Sep 14;43(36):11605-15.

 

Ramadan K, Shevelev IV, Maga G, Hubscher U. De novo DNA synthesis by human DNA polymerase lambda, DNA polymerase mu and terminal deoxyribonucleotidyl transferase. J Mol Biol. 2004 May 28;339(2):395-404.

 

Shevelev I, Blanca G, Villani G, Ramadan K, Spadari S, Hubscher U, Maga G. Mutagenesis of human DNA polymerase lambda: essential roles of Tyr505 and Phe506 for both DNA polymerase and terminal transferase activities. Nucleic Acids Res. 2003 Dec 1;31(23):6916-25.

 

Maga G, Hubscher U. Proliferating cell nuclear antigen (PCNA): a dancer with many partners. J Cell Sci. 2003 Aug 1;116(Pt 15):3051-60. Review.

 

Blanca G, Shevelev I, Ramadan K, Villani G, Spadari S, Hubscher U, Maga G. Human DNA polymerase lambda diverged in evolution from DNA polymerase beta toward specific Mn(++) dependence: a kinetic and thermodynamic study. Biochemistry. 2003 Jun 24;42(24):7467-76.

 

Ramadan K, Maga G, Shevelev IV, Villani G, Blanco L, Hubscher U. Human DNA polymerase lambda possesses terminal deoxyribonucleotidyl transferase activity and can elongate RNA primers: implications for novel functions. J Mol Biol. 2003 Apr 18;328(1):63-72.

 

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 Dec 13;277(50):48434-40. 

 

Hubscher U, Maga G, Spadari S. Eukaryotic DNA polymerases. Annu Rev Biochem. 2002;71:133-63. Epub 2001 Nov 9. Review.

 

Ramadan K, Shevelev IV, Maga G, Hubscher U. DNA polymerase lambda from calf thymus preferentially replicates damaged DNA. J Biol Chem. 2002 May 24;277(21):18454-8.

 

 

 


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