Functional analysis of multiple single-stranded DNA-binding proteins from Methanosarcina acetivorans and their effects on DNA synthesis by DNA polymerase BI.
Single-stranded DNA-binding proteins and their functional homologs, replication protein A, are essential components of cellular DNA replication, repair and recombination. We describe here the isolation and characterization of multiple replication protein A homologs, RPA1, RPA2, and RPA3, from the archaeon Methanosarcina acetivorans. RPA1 comprises four single-stranded DNA-binding domains, while RPA2 and RPA3 are each composed of two such domains and a zinc finger domain. Gel filtration analysis suggested that RPA1 exists as homotetramers and homodimers in solution, while RPA2 and RPA3 form only homodimers. Unlike the multiple RPA proteins found in other Archaea and eukaryotes, each of the M. acetivorans RPAs can act as a distinct single-stranded DNA-binding protein. Fluorescence resonance energy transfer and fluorescence polarization anisotropy studies revealed that the M. acetivorans RPAs bind to as few as 10 single-stranded DNA bases. However, more stable binding is achieved with single-stranded DNA of 18-23 bases, and for such substrates the estimated Kd was 3.82 +/- 0.28 nM, 173.6 +/- 105.17 nM, and 5.92 +/- 0.23 nM, for RPA1, RPA2, and RPA3, respectively. The architectures of the M. acetivorans RPAs are different from those of hitherto reported homologs. Thus, these proteins may represent novel forms of replication protein A. Most importantly, our results show that the three RPAs and their combinations highly stimulate the primer extension capacity of M. acetivorans DNA polymerase BI. Although bacterial SSB and eukaryotic RPA have been shown to stimulate DNA synthesis by their cognate DNA polymerases, our findings provide the first in vitro biochemical evidence for the conservation of this property in an archaeon.
Robbins
Justin B
JB
Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
Murphy
Mary C
MC
White
Bryan A
BA
Mackie
Roderick I
RI
Ha
Taekjip
T
Cann
Isaac K O
IK
D000595:Amino Acid Sequence; D016880:Anisotropy; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D004247:DNA / Q000737:chemistry; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D017020:Methanosarcina / Q000378:metabolism*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D015032:Zinc / Q000737:chemistry; D016335:Zinc Fingers
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D015032:Zinc; D003545:Cysteine