Characterization of the human SNM1A and SNM1B/Apollo DNA repair exonucleases.
Human SNM1A and SNM1B/Apollo have both been implicated in the repair of DNA interstrand cross-links (ICLs) by cellular studies, and SNM1B is also required for telomere protection. Here, we describe studies on the biochemical characterization of the SNM1A and SNM1B proteins. The results reveal some fundamental differences in the mechanisms of the two proteins. Both SNM1A and SNM1B digest double-stranded and single-stranded DNA with a 5'-to-3' directionality in a reaction that is stimulated by divalent cations, and both nucleases are inhibited by the zinc chelator o-phenanthroline. We find that SNM1A has greater affinity for single-stranded DNA over double-stranded DNA that is not observed with SNM1B. Although both proteins demonstrate a low level of processivity on low molecular weight DNA oligonucleotide substrates, when presented with high molecular weight DNA, SNM1A alone is rendered much more active, being capable of digesting kilobase-long stretches of DNA. Both proteins can digest past ICLs induced by the non-distorting minor groove cross-linking agent SJG-136, albeit with SNM1A showing a greater capacity to achieve this. This is consistent with the proposal that SNM1A and SNM1B might exhibit some redundancy in ICL repair. Together, our work establishes differences in the substrate selectivities of SNM1A and SNM1B that are likely to be relevant to their in vivo roles and which might be exploited in the development of selective inhibitors.
Department of Oncology, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK.
Blanka
B
Sengerová
Charles K
CK
Allerston
Mika
M
Abu
Sook Y
SY
Lee
Janet
J
Hartley
Konstantinos
K
Kiakos
Christopher J
CJ
Schofield
John A
JA
Hartley
Opher
O
Gileadi
Peter J
PJ
McHugh
D018797:Cell Cycle Proteins; D002614:Chelating Agents / Q000737:chemistry; D004247:DNA / Q000737:chemistry; D053837:DNA Cleavage; D004249:DNA Damage; D045643:DNA Repair Enzymes / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D004277:DNA, Single-Stranded / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D057075:Enzyme Assays; D004791:Enzyme Inhibitors / Q000737:chemistry; D004926:Escherichia coli; D005090:Exodeoxyribonucleases; D019793:Fluorescein / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008274:Magnesium / Q000737:chemistry; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D010957:Plasmids / Q000737:chemistry; D011485:Protein Binding; D012313:RNA / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013379:Substrate Specificity
D018797:Cell Cycle Proteins; D002614:Chelating Agents; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D012313:RNA; D004247:DNA; C115339:DCLRE1A protein, human; C493741:DCLRE1B protein, human; D005090:Exodeoxyribonucleases; D045643:DNA Repair Enzymes; D008274:Magnesium; D019793:Fluorescein