A Single-Molecule Atomic Force Microscopy Study of PARP1 and PARP2 Recognition of Base Excision Repair DNA Intermediates

The contribution of PARP1, PARP2 and poly(ADP-ribosyl)ation to base excision repair in the nucleosomal context

“…The behaviour of PARP2 under the interaction with the substrates was more complicated (Table 1). PARP2 manifested extremely high affinity for gapped DNA that is in line with previously published findings (31, 55). Additionally strong increase of PARP2 affinity for gapped NCP may suggest a significant contribution of PARP2’s interaction with the histone core of an NCP.…”

“…The activity of both proteins toward the analysed DNA with a given type of damage correlated with these proteins’ affinity, though the activity of PARP2 was always lower as compared to PARP1. Additionally, in search of a possible specific role of PARP1 and PARP2, the interactions of these proteins with DNA structures mimicking various stages BER intermediates have been investigated by AFM (31). In that study, the AFM data indicated that PARP1 has higher affinity for early BER intermediates containing an AP site or a gap with a 5′-deoxyribose phosphate than PARP2 does, whereas PARP2 interacts more efficiently with a 5′-phosphorylated nick and may contribute to the regulation of the final ligation step.…”

“…It was obtained that the k obs values after PARP1 PARylation were dependent on the damage type and were higher for gap-NCP at the comparatively equal K M values (Table 2). This variation may reflect the activation efficacy of PARP1 in the presence of undamaged DNA or various types of DNA damage: the AP site or gap (25, 31, 55). The existence of a specific damage site in the DNA in addition to a blunt end could result in binding of additional PARP1 molecules to the lesion.…”

“…Moreover, the interactions of PARP2 with BER proteins XRCC1, Polβ and LigIIIα have been demonstrated in HeLa cells (33). Recently, the specificity of the interaction of PARP1 and PARP2 with various BER intermediates was analysed by atomic force microscopy (AFM) using long DNA duplex substrates, and the data speak in favour of PARP2 contribution to the later stages of BER (31). Nevertheless, the final function of PARP2 in this process is not clear.…”

PEGylated surfaces for the study of DNA–protein interactions by atomic force microscopy

“…44 Previous AFM studies on PARP-DNA binding, carried out on dried samples, have shown that the sample preparation for observing DNA-bound PARP is non-trivial, and have visualized PARP bound to DNA breaks/ ends as well as to undamaged DNA. [45][46][47] Here we use PARP1 to demonstrate the wider applicability of our method, imaging DNA before and after the addition of the enzyme in solution (Fig. 6).…”