8-oxo-guanine bypass by human DNA polymerases in the presence of auxiliary proteins.
Specialized DNA polymerases (DNA pols) are required for lesion bypass in human cells. Auxiliary factors have an important, but so far poorly understood, role. Here we analyse the effects of human proliferating cell nuclear antigen (PCNA) and replication protein A (RP-A) on six different human DNA pols--belonging to the B, Y and X classes--during in vitro bypass of different lesions. The mutagenic lesion 8-oxo-guanine (8-oxo-G) has high miscoding potential. A major and specific effect was found for 8-oxo-G bypass with DNA pols lambda and eta. PCNA and RP-A allowed correct incorporation of dCTP opposite a 8-oxo-G template 1,200-fold more efficiently than the incorrect dATP by DNA pol lambda, and 68-fold by DNA pol eta, respectively. Experiments with DNA-pol-lambda-null cell extracts suggested an important role for DNA pol lambda. On the other hand, DNA pol iota, together with DNA pols alpha, delta and beta, showed a much lower correct bypass efficiency. Our findings show the existence of an accurate mechanism to reduce the deleterious consequences of oxidative damage and, in addition, point to an important role for PCNA and RP-A in determining a functional hierarchy among different DNA pols in lesion bypass.
Maga
Giovanni
G
Institute of Molecular Genetics IGM-CNR, via Abbiategrasso 207, I-27100 Pavia, Italy. maga@igm.cnr.it
Villani
Giuseppe
G
Crespan
Emmanuele
E
Wimmer
Ursula
U
Ferrari
Elena
E
Bertocci
Barbara
B
Hübscher
Ulrich
U
D000818:Animals; D004249:DNA Damage; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000145:classification / Q000378:metabolism*; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D003845:Deoxycytosine Nucleotides / Q000378:metabolism; D005347:Fibroblasts; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D051379:Mice; D010084:Oxidation-Reduction; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D051716:Replication Protein A / Q000378:metabolism*; D013379:Substrate Specificity; D013698:Templates, Genetic
D003838:Deoxyadenine Nucleotides; D003845:Deoxycytosine Nucleotides; D018809:Proliferating Cell Nuclear Antigen; C497167:RPA1 protein, human; D051716:Replication Protein A; C024107:2'-deoxycytidine 5'-triphosphate; C024829:8-hydroxyguanine; D006147:Guanine; D004259:DNA-Directed DNA Polymerase; C026600:2'-deoxyadenosine triphosphate