Construction of a vector containing a site-specific DNA double-strand break with 3-phosphoglycolate termini and analysis of the products of end-joining in CV-1 cells '

Abstract

Previous studies have shown that linearized SV40-based shuttle vectors transfected into mammalian cells are efficiently recircularized by an error-prone end-joining pathway. To determine whether and with what specificity free radical-mediated double-strand breaks are rejoined by this pathway, a structural mimic of such a break was introduced at a specific site in an SV40-based shuttle vector, by ligating purified 3'-phosphoglycolate-terminated oligonucleotides into 3' recessed ends generated in the linearized vector. These terminally blocked linear vectors were efficiently repaired and replicated when transfected into simian CV-1 cells. Sequencing across the repair joints in progeny plasmid indicated that, for a blunt-ended vector, the most frequent mechanism of rejoining was splicing at a terminal 4-base homology; however, a significant fraction of the joints retained all bases from both ends of the break, consistent with a mechanism involving simple 3'-phosphoglycolate removal, followed by blunt-end ligation. For the analogous 3'-hydroxyl terminated break, the fraction of simple blunt-end ligations was considerably higher. For a phosphoglycolate-terminated vector with cohesive ends the most frequent repair mechanism was simple ligation of the annealed cohesive ends, presumably preceded by phosphoglycolate removal. For all these substrates, the remaining repair joints showed small or large deletions from one or both of the ends, usually with apparent annealing at short (1-4-base) homologies. The results suggest that while breaks with 3'-phosphoglycolates can be repaired, these blocked termini represent a significant barrier to DNA end-joining, and can significantly alter its specificity. The presence of cohesive ends appears to improve markedly the fidelity of rejoining for terminally blocked double-strand breaks.

Loop 1 modulates the fidelity of DNA polymerase

“…To generate internally labeled plasmid substrates, plasmid pRZ56 was linearized with MluI and then 10- and 11-base 5′-overhangs were formed by controlled 3′-resection with T4 DNA polymerase in the presence of dTTP (24). Construction of a vector containing a site-specific DNA double-strand break with 3′-phosphoglycolate termini and analysis of the products of end joining in CV-1 cells (24).…”

“…Construction of a vector containing a site-specific DNA double-strand break with 3′-phosphoglycolate termini and analysis of the products of end joining in CV-1 cells (24). An unlabeled 13-mer (5′pGCCGGACGCGACG) and a 5′-end-labeled 14-mer (5′*pCGAGGAACGCGACG) were ligated into the 10- and 11-base overhangs, respectively, and the plasmid was purified by agarose gel electrophoresis and electroeluted (25).…”

Coordinate 5′ and 3′ endonucleolytic trimming of terminally blocked blunt DNA double-strand break ends by Artemis nuclease and DNA-dependent protein kinase

“…The modified oligomer was purified by gel electrophoresis and HPLC, and its structure verified by mass spectrometry (14). Internally labeled blunt-ended plasmid substrates were prepared by ligating this 5′- 32 P-labeled 11-mer, or an analogous 3′-hydroxyl 11-mer, into an 11-base 5′ overhang of plasmid pRZ56 and were purified by agarose gel electrophoresis as described (15,16). A substrate of identical sequence but with 3′-end label was generated by cutting circular pRZ56 with MluI, and filling in the 4-base 5′ overhang with dGTP and [α- 32 P]dCTP (3000 Ci/mmole, Perkin-Elmer), using exonuclease-deficient Klenow fragment.…”

Phosphorylation in the serine/threonine 2609–2647 cluster promotes but is not essential for DNA-dependent protein kinase-mediated nonhomologous end joining in human whole-cell extracts

“…Previous end-joining experiments employed an SV40-based shuttle plasmid (pSV56) designed to introduce a DSB in a polylinker in the intron of the T-antigen gene (9). However, because the presence of the SV40 origin reduces the yield of the plasmid from bacterial cultures, the polylinker region of pSV56 was excised as a 625-bp PflMI/AvrII fragment, and cloned into pBR322 between the EcoRV and NheI sites (the PflMI cut was blunt-ended with T4 polymerase).…”

“…This procedure resects each 3′-terminal strand to the first thymine in the sequence, resulting in a 10-base 5′ overhang at one end and an 11-base 5′ overhang at the other. An unlabeled 13-mer and a 5′- 32 P-labeled 14-mer, each complementary to one of the 5′ overhangs, were successively ligated into the overhangs, to yield site-specifically labeled substrates with partially complementary (-ACG) overhangs, as described previously (9,10). Control experiments showed that the oligomers were ligated onto at least 90% of the overhangs (data not shown).…”