Abstract
Poly(ADP-ribose) polymerase 1 (PARP1) is both a first responder to DNA damage and a chromatin architectural protein. How PARP1 rapidly finds DNA damage sites in the context of a nucleus filled with undamaged DNA, to which it also binds, is an unresolved question. Here, we show that PARP1 association with DNA is diffusion-limited, and release of PARP1 from DNA is promoted by binding of an additional DNA molecule that facilitates a 'monkey bar' mechanism, also known as intersegment transfer. The WGR-domain of PARP1 is essential to this mechanism, and a point mutation (W589A) recapitulates the altered kinetics of the domain deletion. Demonstrating the physiological importance of the monkey bar mechanism for PARP1 function, the W589A mutant accumulates at sites of DNA damage more slowly following laser micro-irradiation than wild-type PARP1. Clinically relevant inhibitors of PARP1 did not alter the rate or mechanism of the release of PARP1 from DNA.
Highlights
Poly(ADP-ribose) polymerase 1 (PARP1) serves as a first responder to DNA damage and is the founding member and most abundant representative of the large family of diphtheria toxin-like ADP-ribosyltransferases (ARTDs) (Bai, 2015; Bock and Chang, 2016; Beck et al, 2014; Daniels et al, 2015; De Vos et al, 2012; Mashimo et al, 2014; Morales et al, 2014)
We have previously shown that PARP1 serves as a chromatin architectural protein and interacts tightly (Kd ~nM) with and is activated by various nucleosome constructs (Clark et al, 2012; Muthurajan et al, 2014)
Under idealized experimental conditions wherein the concentration of PARP1 greatly exceeds the concentration of p18mer*, one would expect a replot of kobs vs. the concentration of PARP1 to yield a straight line, as was observed here (Figure 2A, inset)
Summary
Poly(ADP-ribose) polymerase 1 (PARP1) serves as a first responder to DNA damage and is the founding member and most abundant representative of the large family of diphtheria toxin-like ADP-ribosyltransferases (ARTDs) (Bai, 2015; Bock and Chang, 2016; Beck et al, 2014; Daniels et al, 2015; De Vos et al, 2012; Mashimo et al, 2014; Morales et al, 2014). Binding to either single or doublestrand DNA breaks (SSBs or DSBs) enzymatically activates PARP1 to use NAD+ in polymerizing long chains of poly(ADP)-ribose (PAR) onto itself and other nuclear acceptor proteins such as histones and DNA repair proteins. Relevant inhibitors of PARP1 bind in the catalytic domain. PARP1 needs to scan for cuts and notches amongst an overwhelming amount of DNA that is still intact This is a complicated task, especially since the protein tends to bind both broken and unbroken DNA. Various models have been put forth and tested for explaining how ‘facilitated diffusion’ could accelerate this search process, all of which recognize the importance, as opposed to hindrance, of non-specific binding to DNA for efficient site localization (Halford and Marko, 2004; Berg et al, 1981; Iwahara et al, 2006; Doucleff and Clore, 2008).
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
