Nucleotide excision repair of a DNA interstrand cross-link produces single- and double-strand breaks

Abstract

The DNA radical resulting from formal abstraction of a hydrogen atom from the thymidine methyl group, 5-(2'-deoxyuridinyl)methyl radical, forms interstrand cross-links with the opposing 2'-deoxyadenosine. This is the first chemically characterized, radical-mediated cross-link between two opposing nucleotides. In addition, cross-linking between opposing bases in the duplex is less common than between those separated by one or two nucleotides. The first step in cross-link repair was investigated using the UvrABC bacterial nucleotide excision repair system. UvrABC incised both strands of the cross-linked DNA, although the strand containing the cross-linked purine was preferred by the enzyme in two different duplexes. The incision sites in one strand were spaced 11-14 nucleotides apart, as is typical for UvrABC incision. The majority of incisions occur at the third phosphate from the 3'-side of the cross-link and eighth or ninth phosphate on the 5'-side. In addition, cleavage was found to occur on both strands, producing double-strand breaks in approximately 25-29% of the incision events. This is the first example of double-strand cleavage during nucleotide excision repair of cross-linked DNA that does not already contain a strand break in the vicinity of the cross-link.

Figure 1

UvrABC incision of 6. A. Strand containing modified thymidine labeled at its 3′-terminus (3′-³²P-“X”-strand) B. Strand containing cross-linked dA labeled at its 3′-terminus (3′-³²P-“Y”-strand) C. Strand containing modified thymidine labeled at its 5′-terminus (5′-³²P-“X”-strand) D. Strand containing cross-linked dA labeled at its 5′-terminus (5′-³²P-“Y”-strand); A + G, sequencing reaction. The cartoons identify the products produced from incision of the unlabeled strand. * indicates position of ³²P-label.

Figure 2

UvrABC incision of 7. A. Strand containing modified thymidine labeled at its 3′-terminus (3′-³²P-“X”-strand) B. Strand containing cross-linked dA labeled at its 3′-terminus (3′-³²P-“Y”-strand) C. Strand containing modified thymidine labeled at its 5′-terminus (5′-³²P-“X”-strand) D. Strand containing cross-linked dA labeled at its 5′-terminus (5′-³²P-“Y”-strand); A + G, sequencing reaction. The cartoons identify the products produced from incision of the unlabeled strand. * indicates position of ³²P-label.

Figure 3

Histograms of UvrABC incision data for: A. 6, B. 7. Arrow length is proportional to relative amounts of incision within one strand.

Figure 4

Identification of UvrABC incision product from 6 on unlabeled strand. A. Independently synthesized postulated products. B. Denaturing PAGE analysis of incision product and independently synthesized standards.

Figure 5

Time course of UvrABC incision of ³²P-6 in which A. 5′-terminus of “X” strand is labeled and B. 5′-terminus of “Y” strand is labeled. ³²P-Labeled terminus is indicated by *. Note: Cartoons of products resulting from incision on the unlabeled strand are shown.

Figure 6

Time course of UvrABC incision of ³²P-7 in which “ A. 5′-terminus of “Y” strand is labeled and B. 5′-terminus of “X” strand is labeled. ³²P-Labeled terminus is indicated by *. Note: Cartoons of products resulting from incision on the unlabeled strand are shown.

Figure 7

Native PAGE analysis of UvrABC incision of 7. A. Strand containing modified thymidine labeled at its 3′-terminus (3′-³²P-“X” strand); B. Strand containing cross-linked dA labeled at its 3′-terminus (3′-³²P-“Y” strand). Compound 5 is used as a single stranded 50 nt marker. 3′-³²P-11 (A) and 3′-³²P-12 (B) are used as double strand cleavage markers. C. Sequences of 11 and 12.

Scheme 1

Interstrand cross-link formation from 5-(2′-deoxyuridinyl)methyl radical (1).

Scheme 2

Interstrand cross-link generation via synthetic precursor.

Scheme 3

Formation of Interstrand cross-links used in this study.

Scheme 4

Repetitive incision of ICL containing radiolabeled “X” strand.

Scheme 5

Repetitive incision of ICL containing radiolabeled “Y” strand.