Bacillus subtilis polynucleotide phosphorylase 3'-to-5' DNase activity is involved in DNA repair.
In the presence of Mn(2+), an activity in a preparation of purified Bacillus subtilis RecN degrades single-stranded (ss) DNA with a 3' --> 5' polarity. This activity is not associated with RecN itself, because RecN purified from cells lacking polynucleotide phosphorylase (PNPase) does not show the exonuclease activity. We show here that, in the presence of Mn(2+) and low-level inorganic phosphate (P(i)), PNPase degrades ssDNA. The limited end-processing of DNA is regulated by ATP and is inactive in the presence of Mg(2+) or high-level P(i). In contrast, the RNase activity of PNPase requires Mg(2+) and P(i), suggesting that PNPase degradation of RNA and ssDNA occur by mutually exclusive mechanisms. A null pnpA mutation (DeltapnpA) is not epistatic with Delta recA, but is epistatic with DeltarecN and Delta ku, which by themselves are non-epistatic. The addA5, Delta recO, Delta recQ (Delta recJ), Delta recU and Delta recG mutations (representative of different epistatic groups), in the context of DeltapnpA, demonstrate gain- or loss-of-function by inactivation of repair-by-recombination, depending on acute or chronic exposure to the damaging agent and the nature of the DNA lesion. Our data suggest that PNPase is involved in various nucleic acid metabolic pathways, and its limited ssDNA exonuclease activity plays an important role in RecA-dependent and RecA-independent repair pathways.
Department of Microbial Biotechnology, Centro Nacional de Biotecnología, CSIC, C/Darwin 3, Campus Universidad Autónoma de Madrid, 28049 Madrid, Spain.
Paula P
PP
Cardenas
Begoña
B
Carrasco
Humberto
H
Sanchez
Gintaras
G
Deikus
David H
DH
Bechhofer
Juan C
JC
Alonso
D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002413:Cations, Divalent; D004260:DNA Repair; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D044127:Epigenesis, Genetic; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D005095:Exoribonucleases / Q000378:metabolism; D008345:Manganese / Q000737:chemistry; D009154:Mutation; D011117:Polyribonucleotide Nucleotidyltransferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D001426:Bacterial Proteins; D002413:Cations, Divalent; D004277:DNA, Single-Stranded; D008345:Manganese; D011117:Polyribonucleotide Nucleotidyltransferase; D005090:Exodeoxyribonucleases; D005095:Exoribonucleases; D004262:DNA Restriction Enzymes; C047879:RecN protein, Bacteria