DNA damage response

Abstract

Structural changes to DNA severely affect its functions, such as replication and transcription, and play a major role in age-related diseases and cancer. A complicated and entangled network of DNA damage response (DDR) mechanisms, including multiple DNA repair pathways, damage tolerance processes, and cell-cycle checkpoints safeguard genomic integrity. Like transcription and replication, DDR is a chromatin-associated process that is generally tightly controlled in time and space. As DNA damage can occur at any time on any genomic location, a specialized spatio-temporal orchestration of this defense apparatus is required.

Figure 1.

Molecular mechanism of nucleotide excision repair (NER). Bulky DNA lesions (e.g., UV-induced photo-products and chemical conjugates to nucleotides) that destabilize the DNA double-helix are targeted by NER. Damage recognition is performed by transcription-coupled NER (TC-NER) and Global Genome NER (GG-NER). It is suggested that prior to damage recognition, chromatin has to be modified. Lesions in the transcribed strand of active genes are detected by the elongating RNA polymerase II (RNAP2) and stabilize the interaction with CSB (step 1b). Within GG-NER, lesions are recognized by the UV-DDB and the XPC complexes (step 1a). These intermediates load transcription factor TFIIH together with the endonuclease XPG (steps 2a and 2b). In TC-NER, CSA is also recruited to modify and reposition lesion-stalled RNAP2 (step 2b). After the two modes of lesion detection, the two processes merge into a common pathway of NER factor assembly by recruiting XPA and replication protein A (RPA) (step 3). This NER-intermediate loads and properly orients the structure-specific endonuclease ERCC1/XPF complex (step 4). After dual incision by XPG (3′ from the lesion) and ERCC1/XPF (5′ from the lesion), a single-strand of 25–29 nucleotides is created (step 5). XPG is likely involved in recruiting the sliding clamp PCNA, which is loaded by RFC and forms the platform for the gap-filling DNA polymerases δ, ε, or κ (step 6). Each of these polymerases has been found to participate in NER-dependent gap-filling. PCNA or RFC are likely also involved in recruiting the ligases (i.e., Ligase I and Ligase III/XRCC1, depending on the proliferation capacity of the cell) to seal the nick (step 7). PCNA also plays a role in attracting the histone-chaperone CAF1 (step 8) to restore the chromatin structure after repair (stage 9).

Figure 2.

Localization of DSBR and NER factors. Typical examples of subnuclear distributions of MDC1, an important factor involved in an early step of DNA double-strand break repair via homologous recombination (A) and XPC, one of the damage-recognizing proteins in NER (B). (A) GFP signal (upper panel) derived from MDC1-GFP stably expressed in U2OS cells, 2 hours after γ-irradiation and fixed with paraformaldehyde, showing the accumulation of MDC1 in typical ionizing-radiation induced foci (IRIF), counter stained with anti-γ-H2AX antibodies (lower panel). (B) NER factors do not accumulate in nuclear foci after DNA damage induction. To allow live cell analysis of NER factor kinetics, local UV-damage infliction through a micro-porous filter is performed (see Fig. 3B,C). XPC-GFP expressed in human fibroblasts (upper panel) accumulates at local UV-damaged sub-nuclear areas as recognized by anti-CPD (the major UV-induced DNA lesion) antibodies (lower panel).

Figure 3.

Live cell analysis of NER. (A) Distribution of three different NER factors tagged with GFP in living cells; DNA is stained by the DNA stain Hoechst. The damage recognition factor XPC is concentrated in nuclear areas (top panel) that also contain high DNA concentrations when the XPC-GFP (Hoogstraten et al. 2008) is expressed in mouse-embryonal fibroblasts that exhibit the species-specific dense DNA-containing areas. This inhomogeneous distribution contrasts to other NER factors such as XPA (Rademakers et al. 2003), which are homogenously distributed (lower panel) and the repair/transcription factor TFIIH that is enriched in nuclei (Hoogstraten et al. 2002). (B) Schematic cartoon of the procedure to locally inflict UV-damage in living cultured cells by irradiation through a microporous filter (Volker et al. 2001). (C) Local accumulation of XPB-GFP (TFIIH subunit) in UV-damaged areas used to determine the dwell time of this NER factor in the damaged area by fluorescence recovery after photo bleaching (FRAP). (D) Human fibroblasts expressing XPC-GFP locally damaged at the indicated position (purple flash, middle panel) by UV-C laser (Dinant et al. 2007). The right panel shows a clear accumulation of this protein as soon as 30 seconds after irradiation. (E) Schematic representation of the quantification of NER factor time-dependent accumulation at local UV-damage in living cells to determine the assembly kinetics of these factors within the chromatin-bound NER complex.