Repair efficiency of clustered abasic sites by APE1 in nucleosome core particles is sequence and position dependent

Abstract

Closely located multiple abasic sites or clustered abasic sites are highly mutagenic and potentially cytotoxic.They have been found to be repair resistant in several in vitro studies. We studied the efficiency of the repair of clustered abasic sites by the APE1 enzyme in nucleosome core particles (NCPs). Sequences having genomic importance as the core sequence of TATA box and CpG islands were used to assemble the NCPs where the abasic clusters are located around the A/T or G/C rich 0.5 positioning site of the NCPs.The thermodynamics of the binding and repair of the A/T or G/C encased clustered abasic sites in the NCPs by APE1 enzyme are reported herein for the first time that was monitored by Isothermal Titration Calorimetry (ITC). The A/T encased clustered abasic sites in the NCP showed greater binding affinity with APE1 than the G/C counterpart. A/T encased abasic sites are also cleaved faster to generate double strand breaks by APE1 enzyme as compared to the CpG island sequence in the NCP, albeit at much slower rate than the linear model. Although, the overall reactivity of the abasic sites is appreciably reduced in the NCPs, distinct differences exist in the processing of the abasic sites that are flanked by A/T or G/C rich sequence. Our study suggests that both sequence effect and nucleosomal positioning are important determinants for the repair efficiency of clustered abasic sites in NCPs. 612 225 7383; Tel: +91 612 255 2057 † Electronic supplementary information (ESI) available. See

Efficient cleavage of single and clustered AP site lesions within mono-nucleosome templates by CHO-K1 nuclear extract contrasts with retardation of incision by purified APE1

“…1 B). At low concentrations of APE1 (<0.4 ng), the level of AP site incision is reduced by 3.6 fold in nucleosomal DNA compared to free DNA, consistent with the reduction reported in [45] . When AP site cleavage becomes saturated in the free DNA (at 0.8 ng APE1) an approximately 4.7 fold decrease was seen in the level of APE1 cleavage of the AP site in nucleosomal-bound DNA.…”

“…Few studies have been undertaken on bistranded clusters in a nucleosome environment. However, two recent studies on bistranded clustered damage showed that the efficiency of glycosylases or APE1 to cause SSB from excision of a lesion within the clusters is even more reduced when in a nucleosome environment and importantly cause suppression of BER-generated DSB [44] , [45] than previously reported in ‘free’ DNA [16] , [17] , [18] .…”

Nucleosomal embedding reshapes the dynamics of abasic sites

“…We are currently using the same protocol to explore the dynamics of a NCP in the presence of clustered abasic sites, to rationalize the drastic increase in DPCs formation rates compared to single AP-sites 49 . MD simulations provide important insights into the finely-tuned sequence effects and dynamics of AP-sites within NCP, which are of utmost important to unravel the complicated mechanisms of their processing by DNA repair enzymes 50 .…”

Nucleosomal embedding reshapes the dynamics of abasic sites

“…We are currently using the same protocol to explore the dynamics of NCP in the presence of clustered abasic sites, to rationalize the drastic increase in DPCs formation rates compared to single AP-sites (44). MD simulations provide important insights into the finely-tuned sequence effects and dynamics of AP-sites within NCP, which are of utmost important to unravel the complicated mechanisms of their repair (45).…”