Crystal structure of the 5hmC specific endonuclease PvuRts1I.

Abstract:

:PvuRts1I is a prototype for a larger family of restriction endonucleases that cleave DNA containing 5-hydroxymethylcytosine (5hmC) or 5-glucosylhydroxymethylcytosine (5ghmC), but not 5-methylcytosine (5mC) or cytosine. Here, we report a crystal structure of the enzyme at 2.35 Å resolution. Although the protein has been crystallized in the absence of DNA, the structure is very informative. It shows that PvuRts1I consists of an N-terminal, atypical PD-(D/E)XK catalytic domain and a C-terminal SRA domain that might accommodate a flipped 5hmC or 5ghmC base. Changes to predicted catalytic residues of the PD-(D/E)XK domain or to the putative pocket for a flipped base abolish catalytic activity. Surprisingly, fluorescence changes indicative of base flipping are not observed when PvuRts1I is added to DNA substrates containing pyrrolocytosine in place of 5hmC (5ghmC). Despite this caveat, the structure suggests a model for PvuRts1I activity and presents opportunities for protein engineering to alter the enzyme properties for biotechnological applications.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Kazrani AA,Kowalska M,Czapinska H,Bochtler M

doi

10.1093/nar/gku186

subject

Has Abstract

pub_date

2014-05-01 00:00:00

pages

5929-36

issue

9

eissn

0305-1048

issn

1362-4962

pii

gku186

journal_volume

42

pub_type

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