Human replication protein A can suppress the intrinsic in vitro mutator phenotype of human DNA polymerase lambda.
DNA polymerase lambda (pol lambda) is a member of the X family DNA polymerases and is endowed with multiple enzymatic activities. In this work we investigated the in vitro miscoding properties of full-length, human pol lambda either in the absence or in the presence of the human auxiliary proteins proliferating cell nuclear antigen (PCNA) and replication protein A (RP-A). Our data suggested that (i) pol lambda had an intrinsic ability to create mismatches and to incorporate ribonucleotides at nearly physiological Mn++ and Mg++ concentrations; (ii) the sequence of the template-primer could influence the misincorporation frequency of pol lambda; (iii) pol lambda preferentially generated G:T and G:G mismatches; (iv) RP-A, but not PCNA, selectively prevented misincorporation of an incorrect nucleotide by pol lambda, without affecting correct incorporation and (v) this inhibitory effect required a precise ratio between the concentrations of pol lambda and RP-A. Possible physiological implications of these findings for the in vivo fidelity of pol lambda are discussed.
Maga
Giovanni
G
Istituto di Genetica Molecolare, IGM-CNR, Pavia, Italy. maga@igm.cnr.it
Shevelev
Igor
I
Villani
Giuseppe
G
Spadari
Silvio
S
Hübscher
Ulrich
U
D020137:Base Pair Mismatch; D019951:DNA Polymerase beta / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000378:metabolism; D006801:Humans; D008274:Magnesium / Q000737:chemistry; D008345:Manganese / Q000737:chemistry; D010641:Phenotype; D018809:Proliferating Cell Nuclear Antigen / Q000502:physiology; D051716:Replication Protein A / Q000502:physiology*; D012265:Ribonucleotides / Q000378:metabolism; D013698:Templates, Genetic
D003854:Deoxyribonucleotides; D018809:Proliferating Cell Nuclear Antigen; C497167:RPA1 protein, human; D051716:Replication Protein A; D012265:Ribonucleotides; D008345:Manganese; C415367:DNA polymerase beta2; D019951:DNA Polymerase beta; D008274:Magnesium