![]() The mutant enzyme retains 5′→3′ exonuclease activity. ( A) A single-strand 3′-Cy5-labeled 25-mer (2 nM) with either a 5′-phosphate or 5′-hydroxyl terminus, was treated with 180 nM wild-type or D165N mutant Artemis for the indicated times and products were analyzed on a 20% sequencing gel. Presence of 5′→3′ exonuclease activity and lack of 5′ and 3′ endonucleolytic trimming activities in Artemis mutant D165N. ( E) Same as (D), except samples were directly loaded on a 36% gel without XbaI treatment. ( D) A substrate with identical terminal sequence and structure, but labeled 25 bases from the 5′-terminus, was similarly treated with Artemis or Artemis/DNA-PK and then cut with XbaI to release terminal fragments. The Artemis-independent 10-mer was not included in the calculations. The total of 2-, 3-, 4-, 5- and 6-base 3′ trimming with T4 pol (filled square), 6-base trimming alone with T4 pol (filled circle) and the total of 2-, 3- and 4-base trimming without T4 pol (open circle), were calculated from two independent experiments error bars show the range of values. ( C) Comparison of 3′ trimming by Artemis/DNA-PK with and without subsequent treatment with T4 pol. Error bars indicate standard errors when larger than the symbols. Abundance of 7- (filled triangle), 8- (filled square) and 9-base (open circle) fragments (corresponding to trimming of 4, 3 and 2 bases, respectively) were determined from three independent experiments similar to that shown in (A). Note that the 10-mer band was present in all T4 pol-treated samples even with Artemis (lane 2) it probably represents an equilibrium between the exonuclease and polymerase functions of the enzyme at the blunt end. ( A) The substrate shown was treated with 25 nM Ku, 50 nM DNA-PKcs and/or 90 nM Artemis for the times indicated, and then half of each sample was treated with T4 pol. Trimming of a 3′-hydroxyl 5′-phosphate substrate by Artemis/DNA-PK. Protein concentrations are the same as in (B). A ‘(+)’ indicates that a component was present in stage 2 because it had been added in stage 1. Artemis and/or KU57788 was then added and the sample incubated for an additional 45 min. ![]() In stage 1, samples were treated with Ku, DNA-PKcs and/or Artemis in the presence or absence of 1 μM KU57788 or 2% DMSO for 30 min. ( E) Effect of the DNA-PKcs inhibitor KU57788 on 3′ and 5′ trimming by Artemis in a two-stage reaction. ( D) Comparison of trimming before and after T4 pol treatment different symbols indicate six independent experiments similar to that shown in (B). ( C) Time course for accumulation of 3′-trimmed 7-, 8- and 9-base fragments without T4 pol treatment (filled circle) or 5-base fragment with T4 pol treatment (open triangle). Following deproteinization, half of each sample was treated with T4 pol, and then all samples were treated with TaqI. Samples were treated for the indicated times with 25 nM Ku, 50 nM DNA-PKcs and/or 90 nM Artemis. ( B) Time course and requirements for 3′ and concomitant 5′ trimming. If 3′ trimming occurs without 5′ trimming (right pathway), T4 pol will fill in the gap, resulting in an increase in labeled 11-base 3′-hydroxyl fragments. ![]() If 3′ trimming is accompanied by 5′ trimming (left pathway), subsequent treatment with T4 pol will resect or fill in the top strand to match the bottom strand. Such processing could account for the very small deletions often found at DNA double-strand break repair sites.Ĭoordinate 5′ and 3′ trimming of a blunt 3′-PG (filled circle) 5′-phosphate substrate by Artemis/DNA-PK. Thus, Artemis and DNA-PK can convert terminally blocked DNA ends of diverse geometry and chemical structure to a form suitable for polymerase-mediated patching and ligation, with minimal loss of terminal sequence. The results suggest that autophosphorylated DNA-PK suppresses the exonuclease activity of Artemis toward blunt-ended DNA, and promotes slow and limited endonucleolytic trimming of the 5'-terminal strand, resulting in short 3' overhangs that are trimmed endonucleolytically. For a blunt end with either a 3'-phosphoglycolate or 3'-hydroxyl terminus, endonucleolytic trimming of 2-4 nucleotides from the 3'-terminal strand was accompanied by trimming of 6 nt from the 5'-terminal strand. In the presence of DNA-PK, Artemis-mediated trimming was more limited, was ATP-dependent and did not require a 5'-phosphate. This resection required a 5'-phosphate, but did not require ATP, and was accompanied by endonucleolytic cleavage of the resulting 3' overhang. In the absence of DNA-PK, Artemis catalyzed extensive 5'->3' exonucleolytic resection of double-stranded DNA. To examine the trimming reaction in more detail, long internally labeled DNA substrates were treated with Artemis. Previous work showed that, in the presence of DNA-dependent protein kinase (DNA-PK), Artemis slowly trims 3'-phosphoglycolate-terminated blunt ends.
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