The 5'-exonuclease activity of bacteriophage T4 RNase H is stimulated by the T4 gene 32 single-stranded DNA-binding protein, but its flap endonuclease is inhibited.
Bacteriophage T4 RNase H is a 5'- to 3'-nuclease that has exonuclease activity on RNA.DNA and DNA.DNA duplexes and can remove the pentamer RNA primers made by the T4 primase-helicase (Hollingsworth, H. C., and Nossal, N. G. (1991) J. Biol. Chem. 266, 1888-1897; Hobbs, L. J., and Nossal, N. G. (1996) J. Bacteriol. 178, 6772-6777). Here we show that this exonuclease degrades duplex DNA nonprocessively, releasing a single oligonucleotide (nucleotides 1-4) with each interaction with the substrate. Degradation continues nonprocessively until the enzyme stops 8-11 nucleotides from the 3'-end of the substrate. T4 gene 32 single-stranded DNA-binding protein strongly stimulates the exonuclease activity of T4 RNase H, converting it into a processive nuclease that removes multiple short oligonucleotides with a combined length of 10-50 nucleotides each time it binds to the duplex substrate. 32 protein must bind on single-stranded DNA behind T4 RNase H for processive degradation. T4 RNase H also has a flap endonuclease activity that cuts preferentially on either side of the junction between single- and double-stranded DNA in flap and fork DNA structures. In contrast to the exonuclease, the endonuclease is inhibited completely by 32 protein binding to the single strand of the flap substrate. These results suggest an important role for T4 32 protein in controlling T4 RNase H degradation of RNA primers and adjacent DNA during each lagging strand cycle.
Bhagwat
M
M
Laboratory of Molecular and Cellular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-0830, USA.
Hobbs
L J
LJ
Nossal
N G
NG
D017122:Bacteriophage T4 / Q000201:enzymology*; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000037:antagonists & inhibitors*; D045585:Flap Endonucleases; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D011485:Protein Binding; D012367:RNA, Viral / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism*; D013379:Substrate Specificity; D014764:Viral Proteins / Q000378:metabolism*
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D012367:RNA, Viral; D014764:Viral Proteins; C060035:gp32 protein, Enterobacteria phage T4; D004706:Endodeoxyribonucleases; D045585:Flap Endonucleases; D016914:Ribonuclease H; D010727:Phosphoric Diester Hydrolases; D043264:Phosphodiesterase I