Abasic sites (Ap site, from apurinic/apirimidinic) are one of the most common lesions generated in DNA by sponaneous base loss or DNA repair processes. There are two equalibrating conformations of Ap site – ring-open aldehyde and cyclic hemiacetal. Ring-opened aldehydes are electrophilic funcional groups capable of formation covalent aduct with nucleophilic sites in DNA. DNA interstrand cross-link (ICL) is a lesion resulting from Ap sites by spontaneous formation of covalent bond between ring-open aldehyde and amin group of adenin residue in the opposite strand of double stranded DNA. ICLs block DNA replication and transcription. The formation of Ap site derived ICL is relatively long process taking several hours[1]. We presume that the ring-openig of an abasic site is the rate-limiting step in the formation of the thermodynamic ICL. There are two types of ICLs. The ratios and the yields of the ICLs are highly depend upon a local sequence[2]. Here we have set up mechanistic experiments in vitro to reveal and calculate the probability of Ap-ICl formation in vivo. In more detail we work on characterization of rates of formation of Ap-ICLs in dependence of sequence of neighbouring nucleotides in the vicinity of freshly formed covalent bond of ICL. We focus on sequence preference, the influence of AT/ GC rich regions and the length of oligonucleotides.
Our experiments extended the understanding of ICL formation and stability to roughly estimate the occurrence of this lesion in vivo. We proved dependence of ICL formation on the bases near the covalent bond of ICL. We also show that in AT rich regions are yields of ICL much higher than in GC rich regions.
Recently, it has been shown that N-glycosidic bond forming ICL is cleaved by NEIL3 glycosylase[3]. Close orthologue of NEIL3 is Formamidopyrimidine DNA glycosylase (Fpg), a DNA base excision repair enzyme with N-glycosylase activity where it removes a wide range of oxidatively damaged bases or Ap lyase activity where it cleaves both 3’- and 5’-phosphodiester bonds of the resulting apurinic/apyrimidinic site. The focus of this part of the project is the preparation of Ap-ICL and its structural characterization using crystallization in complex with Fpg or NEIL3. In contrast with NEIL3 we show that Fpg binds avidly to double-stranded DNA with ICL. We have crystallized both Fpg and Fpg in complex with Ap-ICL and we have already solved structure of Fpg.