DNA double-strand break repair in cell-free extracts from Ku80-deficient cells: implications for Ku serving as an alignment factor in non-homologous DNA end joining - PubMed (original) (raw)

DNA double-strand break repair in cell-free extracts from Ku80-deficient cells: implications for Ku serving as an alignment factor in non-homologous DNA end joining

E Feldmann et al. Nucleic Acids Res. 2000.

Abstract

Non-homologous DNA end joining (NHEJ) is considered the major pathway of double-strand break (DSB) repair in mammalian cells and depends, among other things, on the DNA end-binding Ku70/80 hetero-dimer. To investigate the function of Ku in NHEJ we have compared the ability of cell-free extracts from wild-type CHO-K1 cells, Ku80-deficient xrs6 cells and Ku80-cDNA-complemented xrs6 cells (xrs6-Ku80) to rejoin different types of DSB in vitro. While the two Ku80-proficient extracts were highly efficient and accurate in rejoining all types of DNA ends, the xrs6 extract displayed strongly decreased NHEJ efficiency and accuracy. The lack of accuracy is most evident in non-homologous terminus configurations containing 3'-overhangs that abut a 5'-overhang or blunt end. While the sequences of the 3'-overhangs are mostly preserved by fill-in DNA synthesis in the Ku80-proficient extracts, they are always completely lost in the xrs6 extract so that, instead, small deletions displaying microhomology patches at their breakpoints arise. In summary, our results are consistent with previous results from Ku-deficient yeast strains and indicate that Ku may serve as an alignment factor that not only increases NHEJ efficiency but also accuracy. Furthermore, a secondary NHEJ activity is present in the absence of Ku which is error-prone and possibly assisted by base pairing interactions.

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Figures

Figure 1

Major pathways of accurate NHEJ as observed in vitro in Xenopus eggs (14,19) and mammalian cells (12,21). (A) DNA ends with anti-parallel 5′- or 3′-PSS form short mismatched overlaps at positions of complementary base pairs (black boxes) which determine the patterns of DNA fill-in synthesis (open and black triangles; 18,19). (B) Sequences of 5′- and 3′-PSS in abutting terminus configurations are preserved by fill-in synthesis (14). While fill-in of 5′-PSS (open triangles) can be primed at the recessed 3′-OH group of the same end, fill-in of 3′-PSS (black triangles) can be primed only at the 3′-OH of the abutting terminus which may be a blunt (bl.) end or a 5′-PSS.

Figure 2

Control of extract quality. Top, kinetics (times in min below each lane) at 25°C of incorporation of [α-32P]dCTP in supercoiled 3 kb ENU-treated (asterisk) and 4.2 kb native plasmid substrates in extracts from CHO-K1 (K1), xrs6 and xrs6-Ku80 (Ku80) cells. Lanes 1–9, ccc and oc forms of the plasmid substrates. Lanes 10–18, the same samples after linearization with _Eco_RI show preferential accumulation of radiolabel in the 3 kb band. Bottom, same lanes as at top but after hybridization to a 32P-labeled plasmid probe for control of DNA content. Shift of ccc bands at time 0 (lanes 1, 4 and 7) results from different levels of positive supercoil induced by EthBr in the gel which is caused by residual negative supercoil (relaxed upon extract treatment) plasmids purified from E.coli.

Figure 3

Efficiency of NHEJ in the different extracts. Kinetics (times in min) at 25°C of ligation (Lig.) of cohesive 5′- (Bam) and 3′-PSS (Pst), and blunt ends (Sma) and NHEJ of non-complementary 5′/5′-PSS (Bam/Asp) in extracts from CHO-K1 (K1), xrs6 and xrs6-Ku80 (Ku80) cells. Terminus configurations are shown at the top of each panel with base matches as white letters. Accurate NHEJ of Bam/Asp is known to involve formation of a mismatched overlap whose nicks are closed by ligation (19). Reaction products as marked on the left side: lin., linear; oc, open circle; ccc, covalently closed circle; M, monomer, 3 kb; Di, dimer, 6 kb; Tri, trimer, 9 kb.

Figure 4

Spectra of junctions generated by DNA end joining in extracts from CHO-K1, xrs6 and xrs6-Ku80 cells. (A) Ligation of cohesive and blunt ends; (B) NHEJ of anti-parallel ends by overlap formation; (C) NHEJ of abutting ends by fill-in. Terminus configurations including flanking double-strand sequences are shown at the top of each panel with complementary bases used in junction formation highlighted by white letters. Junctions are listed below as top strand sequences with vertical lines marking breakpoint (middle) and blunt positions (left and right). Note that only the main junctions are shown while less frequent events are summarized under ‘other junctions’, which comprise all three possible types of interactions (between two PSS, one PSS with the flanking duplex of the other terminus and two flanking duplexes). ‘Larger deletions’ designate junctions not determined (n.d.) due to failure of primer annealing during sequencing. Sequences between broken lines highlight ‘accurate’ junctions of which the ones formed by overlap from Bam/Asp, _Bst_X/_Bst_X and Kpn/Pst contain mismatches that yield two different sequences (boxes with two bases) after segregation in E.coli (18,19). Total numbers of bases deleted from each junction are listed below the open triangles with asterisks marking the ‘accurate’ junction. Underlining indicates a restored restriction site used for quicker analysis (e.g. _Bam_S). Microhomology patches (white letters) found at breakpoints are ascribed arbitrarily to the left end although the actual origin of the affected bases cannot be determined unambiguously. Frequencies of junctions obtained in the different extracts are shown on the right. Σ indicates total numbers of junctions analyzed, bold numbers and underlined percentages frequencies of ‘accurate’ junctions, numbers in brackets the fraction of ‘other junctions’ displaying microhomologies of 1–3 bp at their breakpoints.

Figure 4

Figure 5

Features of the Ku-dependent and -independent pathway of NHEJ. In the Ku-dependent pathway, the Ku-heterodimers (grey circles) bind to the ends of a broken DNA duplex (black lines) and serve as alignment factors to mediate, together with DNA-PKCS, XRCC4 and ligase IV, accurate NHEJ that forms both circles and linear dimers. The Rad50/Mre11/NBS1 nuclease complex (in yeast NBS1 is Xrs2) is probably also involved in this pathway (4). In the error-prone Ku-independent pathway, microhomology patches (black boxes) are used for SSA that forms mainly dimers containing deletions. The factors involved in this pathway are presently unknown but the Rad52 protein, one of the key players in homologous recombination, might be a possible candidate because it binds, like Ku, to DNA ends and promotes SSA (3).

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DNA double-strand break repair in cell-free extracts from Ku80-deficient cells: implications for Ku serving as an alignment factor in non-homologous DNA end joining - PubMed (original) (raw)