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Repair of laser-localized DNA interstrand cross-links in G1 phase mammalian cells.

ABSTRACT: Interstrand cross-links (ICLs) are absolute blocks to transcription and replication and can provoke genomic instability and cell death. Studies in bacteria define a two-stage repair scheme, the first involving recognition and incision on either side of the cross-link on one strand (unhooking), followed by recombinational repair or lesion bypass synthesis. The resultant monoadduct is removed in a second stage by nucleotide excision repair. In mammalian cells, there are multiple, but poorly defined, pathways, with much current attention on repair in S phase. However, many questions remain, including the efficiency of repair in the absence of replication, the factors involved in cross-link recognition, and the timing and demarcation of the first and second repair cycles. We have followed the repair of laser-localized lesions formed by psoralen (cross-links/monoadducts) and angelicin (only monoadducts) in mammalian cells. Both were repaired in G(1) phase by nucleotide excision repair-dependent pathways. Removal of psoralen adducts was blocked in XPC-deficient cells but occurred with wild type kinetics in cells deficient in DDB2 protein (XPE). XPC protein was rapidly recruited to psoralen adducts. However, accumulation of DDB2 was slow and XPC-dependent. Inhibition of repair DNA synthesis did not interfere with DDB2 recruitment to angelicin but eliminated recruitment to psoralen. Our results demonstrate an efficient ICL repair pathway in G(1) phase cells dependent on XPC, with entry of DDB2 only after repair synthesis that completes the first repair cycle. DDB2 accumulation at sites of cross-link repair is a marker for the start of the second repair cycle.

SUBMITTER: Muniandy PA

PROVIDER: S-EPMC2788842 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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Repair of laser-localized DNA interstrand cross-links in G1 phase mammalian cells.

Muniandy Parameswary A PA Thapa Dennis D Thazhathveetil Arun Kalliat AK Liu Su-Ting ST Seidman Michael M MM

The Journal of biological chemistry 20090814 41

Interstrand cross-links (ICLs) are absolute blocks to transcription and replication and can provoke genomic instability and cell death. Studies in bacteria define a two-stage repair scheme, the first involving recognition and incision on either side of the cross-link on one strand (unhooking), followed by recombinational repair or lesion bypass synthesis. The resultant monoadduct is removed in a second stage by nucleotide excision repair. In mammalian cells, there are multiple, but poorly define ...[more]

PMID: 19684342

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Project description:Bifunctional DNA alkylating agents form a diverse assortment of covalent DNA interstrand cross-linked (ICL) structures that are potent cytotoxins. Because it is implausible that cells could possess distinct DNA repair systems for each individual ICL, it is believed that common structural and dynamic features of ICL damage are recognized, rather than specific structural characteristics of each cross-linking agent. Investigation of the structural and dynamic properties of ICLs that might be important for recognition has been complicated by heterogeneous incorporation of these lesions into DNA. To address this problem, we have synthesized and characterized several homogeneous ICL DNAs containing site-specific staggered N4-cytosine-ethyl-N4-cytosine cross-links. Staggered cross-links were introduced in two ways, in a manner that preserves the overall structure of B-form duplex DNA and in a manner that highly distorts the DNA structure, with the goal of understanding how structural and dynamic properties of diverse ICL duplexes might flag these sites for repair. Measurements of base pair opening dynamics in the B-form ICL duplex by (1)H NMR line width or imino proton solvent exchange showed that the guanine base opposite the cross-linked cytosine opened at least 1 order of magnitude more slowly than when in a control matched normal duplex. To a lesser degree, the B-form ICL also induced a decrease in base pair opening dynamics that extended from the site of the cross-link to adjacent base pairs. In contrast, the non-B-form ICL showed extensive conformational dynamics at the site of the cross-link, which extended over the entire DNA sequence. Because DNA duplexes containing the B-form and non-B-form ICL cross-links have both been shown to be incised when incubated in mammalian whole cell extracts, while a matched normal duplex is not, we conclude that intrinsic DNA dynamics is not a requirement for specific damage incision of these ICLs. Instead, we propose a general model in which destabilized ICL duplexes serve to energetically facilitate binding of DNA repair factors that must induce bubbles or other distortions in the duplex. However, the essential requirement for incision is an immobile Y-junction where the repair factors are stably bound at the site of the ICL, and the two DNA strands are unpaired.

Dataset Information

Repair of laser-localized DNA interstrand cross-links in G1 phase mammalian cells.

Publications

Repair of laser-localized DNA interstrand cross-links in G1 phase mammalian cells.

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