Replication of 2-hydroxyadenine-containing DNA and recognition by human MutSalpha.
2-Hydroxyadenine (2-OH-A), a product of DNA oxidation, is a potential source of mutations. We investigated how representative DNA polymerases from the A, B and Y families dealt with 2-OH-A in primer extension experiments. A template 2-OH-A reduced the rate of incorporation by DNA polymerase alpha (Pol alpha) and Klenow fragment (Kf(exo-)). Two Y family DNA polymerases, human polymerase eta (Pol eta) and the archeal Dpo4 polymerase were affected differently. Bypass by Pol eta was very inefficient whereas Dpo4 efficiently replicated 2-OH-A. Replication of a template 2-OH-A by both enzymes was mutagenic and caused base substitutions. Dpo4 additionally introduced single base deletions. Thermodynamic analysis showed that 2-OH-A forms stable base pairs with T, C and G, and to a lesser extent with A. Oligonucleotides containing 2-OH-A base pairs, including the preferred 2-OH-A:T, were recognized by the human MutSalpha mismatch repair (MMR). MutSalpha also recognized 2-OH-A located in a repeat sequence that mimics a frameshift intermediate.
Barone
Flavia
F
Unit of Experimental Carcinogenesis, Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy.
McCulloch
Scott D
SD
Macpherson
Peter
P
Maga
Giovanni
G
Yamada
Masami
M
Nohmi
Takehiko
T
Minoprio
Anna
A
Mazzei
Filomena
F
Kunkel
Thomas A
TA
Karran
Peter
P
Bignami
Margherita
M
D020137:Base Pair Mismatch; D001483:Base Sequence; D002478:Cells, Cultured; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D053843:DNA Mismatch Repair; D004256:DNA Polymerase I / Q000378:metabolism; D019951:DNA Polymerase beta / Q000378:metabolism; D004261:DNA Replication / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006147:Guanine / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D051718:MutS Homolog 2 Protein / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D013696:Temperature; D013816:Thermodynamics; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; C094443:G-T mismatch-binding protein; D006147:Guanine; D004247:DNA; C008183:isoguanine; D004256:DNA Polymerase I; D019951:DNA Polymerase beta; C497172:MSH2 protein, human; D051718:MutS Homolog 2 Protein