Wspólnotowy Serwis Informacyjny Badan i Rozwoju - CORDIS

Propertties of human family X DNA polymerase lambda

- DNA polymerase (pol) lambda is homologous to pol beta and has intrinsic polymerase and terminal transferase activities. However, nothing was known about the amino acid residues involved in these activites. In order to precisely define the nucleotide-binding site of human pol lambda, we have mutagenised two amino acids, Tyr505 and the neighbouring Phe506, which were predicted by structural homology modelling to correspond to the Tyr271 and Phe272 residues of pol beta, which are involved in nucleotide binding. Our analysis demonstrated that pol lambda Phe506Arg/Gly mutants possess very low polymerase and terminal transferase activities as well as greatly reduced abilities for processive DNA synthesis and for carrying on translesion synthesis past an abasic site. The Tyr505Ala mutant, on the other hand, showed an altered nucleotide binding selectivity to perform the terminal transferase activity. Our results suggest the existence of a common nucleotide-binding site for the polymerase and terminal transferase activities of pol lambda, as well as distinct roles of the amino acids Tyr505 and Phe506 in these two catalytic functions.

- Pol lambda and pol mu are ubiquitous enzymes, possess both DNA polymerase and terminal deoxyribonucleotidyl transferase activities and belong to pol X family, together with pol beta and TdT. Here we show that pol lambda, pol mu and TdT, all possess the ability to synthesise in vitro short fragments of DNA in the absence of a primer-template or even a primer or a template in the reaction. The DNA synthesised de novo by pol lambda, pol mu and TdT appears to have an unusual structure. Furthermore we found that the amino acid Phe506 of pol lambda is essential for the de novo synthesis. This novel catalytic activity might be related to the proposed functions of these three pol X family members in DNA repair and DNA recombination.

- Human DNA polymerases (pols) beta and lambda could promote template slippage and generate -1frameshifts on defined heteropolymeric DNA substrates containing a single abasic site. Kinetic data demonstrated that pol ìambda was more efficient than pol beta in catalyzing translesion DNA synthesis past an abasic site, particularly in the presence of low nucleotide concentrations. Moreover, pol lambda was found to generate frameshifts in two ways: first, by using a nucleotide-stabilized primer misalignment mechanism, or second, by promoting primer reannealing using microhomology regions between the terminal primer sequence and the template strand. Our results suggest a molecular mechanism for the observed high in vivo rate of frameshifts generation by pol lambda and highlight the remarkable ability of pol lambda to promote microhomology pairing between two DNA strands, further supporting its proposed role in the nonhomologous end joining process.

- We showed also that DNA polymerase lambda interacts with proliferating cell nuclear antigen (PCNA) in vivo in human cells. Moreover, by using recombinant mutated PCNA, we could demonstrate that pol lambda interacts with both the interdomain-connecting loop and the nearby hydrophobic pocket on the anterior of PCNA and that critical residues within a helix-hairpinhelix domain of pol lambda, important for proper DNA primer binding, are also involved in the enzyme’s interaction with PCNA. Finally, we showed that the tumor suppressor protein p21WAF1/CIP1 can efficiently compete in vitro with pol lambda for binding to PCNA. Given the high rate of frameshift mutations induced by pol lambda and its ability to bypass abasic sites, accurate regulation of pol lambda activity by PCNA and p21 concerted action might be important for preventing genetic instability.

- We enzymologically characterized the terminal transferase activity of polymerase lambda (pol lambda-tdt). The tdt activity was strongly influenced by the nature of the 3-terminal sequence of the DNA substrate, and it required a single-stranded (ss) DNA 3-overhang of about 9 12 nucleotides for optimal activity. The strong preference observed for pyrimidine versus purine nucleotide incorporation was found to be due, at least partially, to a steric block imposed by the residue Tyr-505 in the active site of pol lambda. Pol lambda-tdt was found to be able to elongate a 3-ssDNA end by two alternative mechanisms. Most importantly we found that the PCNA was able to selectively block the looping back mechanism while stimulating the single terminal nucleotide addition. Finally RP-A completely suppressed the transferase activity of pol lambda while stimulating the polymerase activity, suggesting that PCNA and RP-A can coordinate the polymerase and the terminal transferase activities of pol lambda.

Reported by

CNR
IGBE-CNR Via abbiategrasso 207
27100 Pavia
Italy
See on map
Śledź nas na: RSS Facebook Twitter YouTube Zarządzany przez Urząd Publikacji UE W górę