Interstrand DNA cross-links are highly cytotoxic because they prevent strand separation that is required for DNA transcription and replication. We recently reported new types of DNA interstrand cross-links involving reaction of abasic (Ap) sites with adenine and guanine residues on the opposing strand of the duplex. Ap sites are abundant in genomic DNA and Ap-derived cross-links could be important in aging, mutagenesis, and the etiology of human cancers. The formation of Ap-derived cross-links is reversible and it is important to determine whether the cross-links are chemically stable enough to block an advancing DNA polymerase. To shed light on this issue, we employed the highly processive, strand displacing polymerase F29 to examine the stability of the dA-Ap cross-link. Using this polymerase it was possible to embed an Ap-derived cross-link in a duplex and monitor the ability of the polymerase to extend a 32P-labeled primer past the cross-link lesion.  We found that the Ap-derived cross-links were strong blocks to F29 regardless of whether the Ap or the dA residue of the cross-linkage was in the template strand. The major site for stalling of the polymerase was at the base directly preceding the cross-link site. This result provided evidence that the protein tunnel seen in the X-ray crystal structure of F29, and proposed to guide a 5-6 base length of the template strand into the active site, is dynamic and allows the cross-link lesion to approach the polymerase active site. A control experiment showed that an Ap site in the displaced strand had no effect on primer extension by F29. On the other hand, an Ap site in the template strand was a mild block to the polymerase. A benzyl oxime adduct on the template strand presented a stronger block to F29 than the Ap site. The results show that the dA-Ap cross-link is stable against a DNA polymerase and, more generally, reveals that F29 can be a useful tool for the detection and characterization of lesions in duplex DNA.

Blocking of the DNA polymerase F29 by abasic site derived cross-links

248th National Meeting (2014)

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