Gali, H. | bioRxiv Science

bioRxiv2024

A bifunctional antibody conjugate marks the location of DNA binding proteins on deproteinized DNA fibers.

Immunofluorescent foci of DNA Damage Response (DDR) proteins serve as surrogates for DNA damage and are frequently interpreted as denoting specific lesions. For example, Double Strand Breaks (DSBs) are potent inducers of the DDR, whose best-known factor is the phosphorylated histone variant H2AX ({gamma}-H2AX). The association with DSBs is so well established that the reverse interpretation that {gamma}-H2AX invariably implies DSBs is routine. However, this conclusion is inferential and has been challenged. The resolution of this question has been hampered by the lack of methods for distinguishing the location of DDR proteins relative to DSBs caused by sequence indifferent agents. Here, we describe an approach for marking the location of DDR factors in relation to DSBs on DNA fibers. We synthesized a two-arm "Y" conjugate containing biotin and trimethylpsoralen (TMP) coupled to a secondary antibody. After exposure to a DNA breaker, permeabilized mammalian cells were incubated with a primary antibody against the DDR factor followed by binding of the secondary antibody in the conjugate to the primary antibody. Exposure to longwave UV light covalently linked the psoralen to the DNA. DNA fibers were spread, and the immunofluorescence of the biotin tag denoted the location of the target protein. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/609705v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@15eaf5corg.highwire.dtl.DTLVardef@14ade70org.highwire.dtl.DTLVardef@51c83forg.highwire.dtl.DTLVardef@131cb58_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO C_FIG

biochemistry↗

bioRxiv2023

RB loss sensitizes cells to replication-associated DNA damage by PARP inhibition

The retinoblastoma tumor suppressor protein (RB) interacts physically and functionally with a number of epigenetic modifying enzymes to control transcriptional regulation, respond to replication stress, promote DNA damage response and repair pathways, and regulate genome stability. To better understand how disruption of RB function impacts epigenetic regulation of genome stability and determine whether such changes may represent exploitable weaknesses of RB-deficient cancer cells, we performed an imaging-based screen to identify epigenetic inhibitors that promote DNA damage and compromise viability of RB-deficient cells. We found that loss of RB alone leads to high levels of replication-dependent poly-ADP ribosylation (PARylation) and that preventing PARylation through inhibition of PARP enzymes enables RB-deficient cells to progress to mitosis with unresolved replication stress and under-replicated DNA. These defects contribute to high levels of DNA damage, decreased proliferation, and compromised cell viability. We demonstrate this sensitivity is conserved across a panel of inhibitors that target both PARP1 and PARP2 and can be suppressed by re-expression of the RB protein. Together, these data indicate that inhibitors of PARP1 and PARP2 may be clinically relevant for RB-deficient cancers.

cell biology↗