Single molecule detection of PARP1 and PARP2 interaction with DNA strand breaks and their poly(ADP-ribosyl)ation using high-resolution AFM imaging

Maria Sukhanova; Sanae Abrakhi; Vandana Joshi; David Pastre; Mikhail Kutuzov; Rashid Anarbaev; Patrick Curmi; Loic Hamon; Olga Lavrik

Single molecule detection of PARP1 and PARP2 interaction with DNA strand breaks and their poly(ADP-ribosyl)ation using high-resolution AFM imaging

Maria Sukhanova ^{1, 2} Sanae Abrakhi ² Vandana Joshi ² David Pastre ² Mikhail Kutuzov ¹ Rashid Anarbaev ^{1, 3} Patrick Curmi ² Loic Hamon ² Olga Lavrik ^{1, 3}

1 Novosibirsk Institute of Chemical Biology and Fundamental Medecine

2 SABNP - Structure et activité des biomolécules normales et pathologiques

3 NSU - Novosibirsk State University

Abstract : PARP1 and PARP2 are implicated in the synthesis of poly(ADP-ribose) (PAR) after detection of DNA damage. The specificity of PARP1 and PARP2 interaction with long DNA fragments containing single- and/or double-strand breaks (SSBs and DSBs) have been studied using atomic force microscopy (AFM) imaging in combination with biochemical approaches. Our data show that PARP1 localizes mainly on DNA breaks and exhibits a slight preference for nicks over DSBs, although the protein has a moderately high affinity for undamaged DNA. In contrast to PARP1, PARP2 is mainly detected at a single DNA nick site, exhibiting a low level of binding to undamaged DNA and DSBs. The enhancement of binding affinity of PARP2 for DNA containing a single nick was also observed using fluorescence titration. AFM studies reveal that activation of both PARPs leads to the synthesis of highly branched PAR whose size depends strongly on the presence of SSBs and DSBs for PARP1 and of SSBs for PARP2. The initial affinity between the PARP1, PARP2 and the DNA damaged site appears to influence both the size of the PAR synthesized and the time of residence of PARylated PARP1 and PARP2 on DNA damages.

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Journal articles

Domain :

Life Sciences [q-bio]

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Single molecule detection of PARP1 and PARP2 interaction with DNA strand breaks and their poly(ADP-ribosyl)ation using high-resolution AFM imaging

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Single molecule detection of PARP1 and PARP2 interaction with DNA strand breaks and their poly(ADP-ribosyl)ation using high-resolution AFM imaging

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