The 3'-5' proofreading exonuclease of archaeal family-B DNA polymerase hinders the copying of template strand deaminated bases.
Archaeal family B polymerases bind tightly to the deaminated bases uracil and hypoxanthine in single-stranded DNA, stalling replication on encountering these pro-mutagenic deoxynucleosides four steps ahead of the primer-template junction. When uracil is specifically bound, the polymerase-DNA complex exists in the editing rather than the polymerization conformation, despite the duplex region of the primer-template being perfectly base-paired. In this article, the interplay between the 3'-5' proofreading exonuclease activity and binding of uracil/hypoxanthine is addressed, using the family-B DNA polymerase from Pyrococcus furiosus. When uracil/hypoxanthine is bound four bases ahead of the primer-template junction (+4 position), both the polymerase and the exonuclease are inhibited, profoundly for the polymerase activity. However, if the polymerase approaches closer to the deaminated bases, locating it at +3, +2, +1 or even 0 (paired with the extreme 3' base in the primer), the exonuclease activity is strongly stimulated. In these situations, the exonuclease activity is actually stronger than that seen with mismatched primer-templates, even though the deaminated base-containing primer-templates are correctly base-paired. The resulting exonucleolytic degradation of the primer serves to move the uracil/hypoxanthine away from the primer-template junction, restoring the stalling position to +4. Thus the 3'-5' proofreading exonuclease contributes to the inability of the polymerase to replicate beyond deaminated bases.
Institute of Cell and Molecular Biosciences (ICaMB), University of Newcastle, Newcastle upon Tyne, NE2 4HH, UK.
Henry J
HJ
Russell
Tomas T
TT
Richardson
Kieran
K
Emptage
Bernard A
BA
Connolly
D019843:Archaeal Proteins / Q000737:chemistry / Q000378:metabolism*; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D003641:Deamination; D005090:Exodeoxyribonucleases / Q000737:chemistry / Q000378:metabolism*; D019271:Hypoxanthine / Q000737:chemistry / Q000378:metabolism; D020027:Pyrococcus furiosus / Q000201:enzymology*; D013698:Templates, Genetic; D014498:Uracil / Q000737:chemistry / Q000378:metabolism*
D019843:Archaeal Proteins; D017931:DNA Primers; D019271:Hypoxanthine; D014498:Uracil; C072146:Pfu DNA polymerase; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases