PARP1 changes from three-dimensional DNA damage searching to one-dimensional diffusion after auto-PARylation or in the presence of APE1

Abstract

PARP1-dependent poly-ADP-ribosylation (PARylation) participates in the repair of many forms of DNA damage. Here, we used atomic force microscopy (AFM) and single molecule fluorescence microscopy to examine the interactions of PARP1 with common DNA repair intermediates. AFM volume analysis indicates that PARP1 binds to DNA at nicks, abasic (AP) sites, and ends as a monomer. Single molecule DNA tightrope assays were used to follow the real-time dynamic behavior of PARP1 in the absence and presence of AP endonuclease (APE1) on AP DNA damage arrays. These experiments revealed that PARP1 conducted damage search mostly through 3D diffusion. Co-localization of APE1 with PARP1 on DNA was found capable of inducing 1D diffusion of otherwise nonmotile PARP1, while excess APE1 also facilitated the dissociation of DNA-bound PARP1. Moreover, auto-PARylation of PARP1 allowed the protein to switch its damage search strategy by causing a 3-fold increase in linear diffusion. Finally, we demonstrated that PARP inhibitor olaparib did not significantly alter the rate of PARP1 dissociation from DNA, but instead resulted in more motility of DNA-bound PARP1 molecules.

Investigation of the three-dimensional structure and interaction mechanism of poly (ADP-ribose) polymerase 4

“…PARP1 and PARP2 were interacted with NAD ( Figure 12(A, B)). The molecular weights of PARP1 and PARP2 were obtained with electrophoresis and chromatography analysis and were found to be similar to previous reports [25][26][27]. Then they were interfered with NAD and the theoretically determined regions were tested.…”

“…Electrophoresis and chromatographic analysis confirmed the purity of the commercially available PARP1 and PARP2 enzymes to be used in the following stages of the study. Chromatographic analyses of both molecules confirmed their molecular weights (Figure 13(B)) [25][26][27]. These enzymes were then interacted with NAD to proceed to the next step.…”

Viscoelastic subdiffusion in a random Gaussian environment

“…The research field of anomalous diffusion and transport [1][2][3][4][5][6][7][8][9][10][11] currently flourishes getting ever more experimental support and manifestations in such diverse research areas as transport processes in living cells and polymeric solutions , colloidal systems [12,15,43,44], dust plasmas [45], organic photoconductors [46], conformational diffusion in proteins [47][48][49][50][51][52][53][54][55][56], self-diffusion in lipid bilayers [57][58][59], diffusion of proteins on DNA strands [60][61][62], to name just a few. Differently from normal diffusion, α = 1, the variance of the diffusing particle positions, δx 2 (t) ∝ t α , often grows sublinearly, α < 1, or superlinearly, α > 1, in time, with some power law exponent α.…”

Poly(ADP-ribose) polymerase 1 Searches DNA via a ‘Monkey Bar’ Mechanism

“…A recent study of PARP1 using single molecule DNA tightrope assays provides strong evidence for such a monkey bar mechanism 24 . It was shown that micro-dissociation of one of PARP1’s multiple DNA binding domains from DNA allows it to bind to a free 37 bp fragment, thus preventing rebinding of the domain to the tightrope and accelerating overall macro-dissociation.…”

“…Additionally, atomic force microscopy has shown that PARP1 binds not only to DNA ends or specific nicks, but also has significant affinity for undamaged DNA 23 . Most recently, single molecule tightrope assays have demonstrated that PARP1 interacts with and moves along undamaged DNA 24 .…”