title: Single molecule detection of PARP1 and PARP2 interaction with DNA strand breaks and their poly(ADP-ribosyl)ation using high-resolution AFM imaging.
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.
authors.affiliation: Institute of Chemical Biology and Fundamental Medicine, 630090, Novosibirsk, Russian Federation INSERM, U1204, Laboratoire Structure-Activité des Biomolécules Normales et Pathologiques, Université d'Evry-Val-d'Essonne, F-91025 Evry, France.
authors.forename: Maria V
authors.initials: MV
authors.lastname: Sukhanova
authors.affiliation: INSERM, U1204, Laboratoire Structure-Activité des Biomolécules Normales et Pathologiques, Université d'Evry-Val-d'Essonne, F-91025 Evry, France.
authors.forename: Sanae
authors.initials: S
authors.lastname: Abrakhi
authors.affiliation: INSERM, U1204, Laboratoire Structure-Activité des Biomolécules Normales et Pathologiques, Université d'Evry-Val-d'Essonne, F-91025 Evry, France.
authors.forename: Vandana
authors.initials: V
authors.lastname: Joshi
authors.affiliation: INSERM, U1204, Laboratoire Structure-Activité des Biomolécules Normales et Pathologiques, Université d'Evry-Val-d'Essonne, F-91025 Evry, France.
authors.forename: David
authors.initials: D
authors.lastname: Pastre
authors.affiliation: Institute of Chemical Biology and Fundamental Medicine, 630090, Novosibirsk, Russian Federation.
authors.forename: Mikhail M
authors.initials: MM
authors.lastname: Kutuzov
authors.affiliation: Institute of Chemical Biology and Fundamental Medicine, 630090, Novosibirsk, Russian Federation Novosibirsk State University, 630090, Novosibirsk, Russian Federation.
authors.forename: Rashid O
authors.initials: RO
authors.lastname: Anarbaev
authors.affiliation: INSERM, U1204, Laboratoire Structure-Activité des Biomolécules Normales et Pathologiques, Université d'Evry-Val-d'Essonne, F-91025 Evry, France.
authors.forename: Patrick A
authors.initials: PA
authors.lastname: Curmi
authors.affiliation: INSERM, U1204, Laboratoire Structure-Activité des Biomolécules Normales et Pathologiques, Université d'Evry-Val-d'Essonne, F-91025 Evry, France loic.hamon@univ-evry.fr.
authors.forename: Loic
authors.initials: L
authors.lastname: Hamon
authors.affiliation: Institute of Chemical Biology and Fundamental Medicine, 630090, Novosibirsk, Russian Federation Novosibirsk State University, 630090, Novosibirsk, Russian Federation lavrik@niboch.nsc.ru.
authors.forename: Olga I
authors.initials: OI
authors.lastname: Lavrik
mesh_terms: D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D053904:DNA Breaks, Single-Stranded; D004260:DNA Repair; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008274:Magnesium / Q000737:chemistry; D018625:Microscopy, Atomic Force; D057054:Molecular Imaging; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D000071137:Poly (ADP-Ribose) Polymerase-1; D011064:Poly Adenosine Diphosphate Ribose / Q000096:biosynthesis* / Q000235:genetics; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011700:Putrescine / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013095:Spermidine / Q000737:chemistry
chemical_list: D011994:Recombinant Proteins; D011064:Poly Adenosine Diphosphate Ribose; D004247:DNA; C499320:PARP1 protein, human; C483041:PARP2 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D008274:Magnesium; D013095:Spermidine; D011700:Putrescine