PspGI, a type II restriction endonuclease from the extreme thermophile Pyrococcus sp.: structural and functional studies to investigate an evolutionary relationship with several mesophilic restriction enzymes.
We present here the first detailed biochemical analysis of an archaeal restriction enzyme. PspGI shows sequence similarity to SsoII, EcoRII, NgoMIV and Cfr10I, which recognize related DNA sequences. We demonstrate here that PspGI, like SsoII and unlike EcoRII or NgoMIV and Cfr10I, interacts with and cleaves DNA as a homodimer and is not stimulated by simultaneous binding to two recognition sites. PspGI and SsoII differ in their basic biochemical properties, viz. stability against chemical denaturation and proteolytic digestion, DNA binding and the pH, MgCl(2) and salt-dependence of their DNA cleavage activity. In contrast, the results of mutational analyses and cross-link experiments show that PspGI and SsoII have a very similar DNA binding site and catalytic center as NgoMIV and Cfr10I (whose crystal structures are known), and presumably also as EcoRII, in spite of the fact that these enzymes, which all recognize variants of the sequence -/CC-GG- (/ denotes the site of cleavage), are representatives of different subgroups of type II restriction endonucleases. A sequence comparison of all known restriction endonuclease sequences, furthermore, suggests that several enzymes recognizing other DNA sequences also share amino acid sequence similarities with PspGI, SsoII and EcoRII in the region of the presumptive active site. These results are discussed in an evolutionary context.
Pingoud
Vera
V
Institut für Biochemie, Justus-Liebig-Universität, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany. vera.pingoud@chemie.bio.uni-giessen.de
Conzelmann
Charlotte
C
Kinzebach
Steffen
S
Sudina
Anna
A
Metelev
Valeri
V
Kubareva
Elena
E
Bujnicki
Janusz M
JM
Lurz
Rudi
R
Lüder
Gerhild
G
Xu
Shuang-Yong
SY
Pingoud
Alfred
A
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001386:Azides / Q000737:chemistry; D001665:Binding Sites; D020134:Catalytic Domain; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004252:DNA Mutational Analysis; D004262:DNA Restriction Enzymes / Q000737:chemistry / Q000235:genetics*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D019143:Evolution, Molecular; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D008854:Microscopy, Electron / Q000379:methods; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010777:Photochemistry / Q000379:methods; D011489:Protein Denaturation; D019711:Pyrococcus / Q000201:enzymology*; D012492:Salts / Q000737:chemistry; D017386:Sequence Homology, Amino Acid
D019843:Archaeal Proteins; D001386:Azides; D003432:Cross-Linking Reagents; D004220:Disulfides; D009838:Oligodeoxyribonucleotides; D012492:Salts; D004247:DNA; D004262:DNA Restriction Enzymes; C045297:CCNGG-specific type II deoxyribonucleases; C046545:CCWGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; D008274:Magnesium; D003545:Cysteine; C011171:4-azidophenacyl bromide