Automated modeling of protein accumulation at DNA damage sites using qFADD.py.

Eukaryotic cells are constantly subject to DNA damage, often with detrimental consequences for the health of the organism. Cells mitigate this DNA damage through a variety of repair pathways involving a diverse and large number of different proteins. To better understand the cellular response to DNA damage, one needs accurate measurements of the accumulation, retention, and dissipation timescales of these repair proteins.

The BRCT domain of PARP1 binds intact DNA and mediates intrastrand transfer.

PARP1 is a key player in the response to DNA damage and is the target of clinical inhibitors for the treatment of cancers. Binding of PARP1 to damaged DNA leads to activation wherein PARP1 uses NAD to add chains of poly(ADP-ribose) onto itself and other nuclear proteins. PARP1 also binds abundantly to intact DNA and chromatin, where it remains enzymatically inactive.

Bridging of nucleosome-proximal DNA double-strand breaks by PARP2 enhances its interaction with HPF1.

Poly(ADP-ribose) Polymerase 2 (PARP2) is one of three DNA-dependent PARPs involved in the detection of DNA damage. Upon binding to DNA double-strand breaks, PARP2 uses nicotinamide adenine dinucleotide to synthesize poly(ADP-ribose) (PAR) onto itself and other proteins, including histones. PAR chains in turn promote the DNA damage response by recruiting downstream repair factors.

Probing the Conformational Changes Associated with DNA Binding to PARP1.

Poly(ADP-ribose) polymerase 1 (PARP1) is an important first responder in the mechanism of DNA repair in eukaryotic cells. It is also a validated drug target, with four different PARP inhibitors (PARPi) approved for the treatment of BRCA-negative cancers. Despite past efforts, many aspects of PARPi are poorly understood, in particular their ability to trap PARP1 on chromatin and the relationships between their potencies, cellular toxicities, and trapping efficiencies.

Quantitating repair protein accumulation at DNA lesions: Past, present, and future.

All organisms must protect their genome from constantly occurring DNA damage. To this end, cells have evolved complex pathways for repairing sites of DNA lesions, and multiple in vitro and in vivo techniques have been developed to study these processes. In this review, we discuss the commonly used laser microirradiation method for monitoring the accumulation of repair proteins at DNA damage sites in cells, and we outline several strategies for deriving kinetic models from such experimental data.

Q-FADD: A Mechanistic Approach for Modeling the Accumulation of Proteins at Sites of DNA Damage.

The repair of DNA damage requires the ordered recruitment of many different proteins that are responsible for signaling and subsequent repair. A powerful and widely used tool for studying the orchestrated accumulation of these proteins at damage sites is laser microirradiation in live cells, followed by monitoring the accumulation of the fluorescently labeled protein in question. Despite the widespread use of this approach, there exists no rigorous method for characterizing the recruitment process quantitatively.

Poly(ADP-ribose) polymerase 1 searches DNA via a 'monkey bar' mechanism.

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.

Efficient differentiation of murine embryonic stem cells requires the binding of CXXC finger protein 1 to DNA or methylated histone H3-Lys4.

Mammalian CXXC finger protein 1 (Cfp1) is a DNA-binding protein that is a component of the Setd1 histone methyltransferase complexes and is a critical epigenetic regulator of both histone and cytosine methylation. Murine embryonic stem (ES) cells lacking Cfp1 exhibit a loss of histone H3-Lys4 tri-methylation (H3K4me3) at many CpG islands, and a mis-localization of this epigenetic mark to heterochromatic sub-nuclear domains. Furthermore, these cells fail to undergo cellular differentiation in vitro.