Analysis of the DNA damage response in living cells

DNA lesions arising from environmental and endogenous sources
induce various cellular responses including cell cycle arrest, DNA
repair and apoptosis. Although detailed insights into the
biochemical mechanisms and composition of DNA repair pathways have
been obtained from in vitro experiments, a better understanding of
the interplay and regulation of these pathways requires DNA repair
studies in living cells. In this study we employed laser
microirradiation and photobleaching techniques in combination with
specific mutants and inhibitors to analyze the real-time
accumulation of proteins at laser-induced DNA damage sites in vivo,
thus unravelling the mechanisms underlying the coordination of DNA
repair in living cells. The immediate and faithful recognition of
DNA lesions is central to cellular survival, but how these lesions
are detected within the context of chromatin is still unclear. In
vitro data indicated that the DNA-damage dependent poly(ADP-ribose)
polymerases, PARP-1 and PARP-2, are involved in this crucial step
of DNA repair. With specific inhibitors, mutations and
photobleaching analysis we could reveal a complex feedback
regulated mechanism for the recruitment of the DNA damage sensor
PARP-1 to microirradiated sites. Activation of PARP-1 results in
localized poly(ADP-ribosyl)ation and amplifies a signal for the
subsequent rapid recruitment of the loading platform XRCC1 which
coordinates the assembly of the repair machinery. Using similar
techniques we could demonstrate the immediate and transient binding
of the RNA Polymerase II cofactor PC4 to DNA damage sites, which
depended on its single strand binding capacity. This establishes an
interesting link between DNA repair and transcription. We propose a
role for PC4 in the early steps of the DNA damage response,
recognizing and stabilizing single stranded DNA (ssDNA) and thereby
facilitating DNA repair by enabling repair factors to access their
substrates. After DNA lesions have been successfully detected they
have to be handed over to the repair machinery which restores
genome integrity. Efficient repair requires the coordinated
recruitment of multiple enzyme activities which is believed to be
controlled by central loading platforms. As laser microirradiation
induces a variety of different DNA lesions we could directly
compare the recruitment kinetics of the two loading platforms PCNA
and XRCC1 which are involved in different repair pathways side by
side. We could demonstrate that PCNA and XRCC1 show distinct
recruitment and binding kinetics with the immediate and fast
recruitment of XRCC1 preceding the slow and continuous recruitment
of PCNA. Introducing consecutively multiple DNA lesions within a
single cell, we further demonstrated that these different
recruitment and binding characteristics have functional
consequences for the capacity of PCNA and XRCC1 to respond to
successive DNA damage events. To further study the role of PCNA and
XRCC1 as loading platforms in DNA repair, we extended our analysis
to their respective interaction partners DNA Ligase I and III.
Although these DNA Ligases are highly homologous and catalyze the
same enzymatic reaction, they are not interchangeable and fulfil
unique functions in DNA replication and repair. With deletion and
mutational analysis we could identify domains mediating the
specific recruitment of DNA Ligase I and III to distinct repair
pathways through their interaction with PCNA and XRCC1. We conclude
that this specific targeting may have evolved to accommodate the
particular requirements of different repair pathways (single
nucleotide replacement vs. synthesis of short stretches of DNA) and
thus enhances the efficiency of DNA repair. Interestingly, we found
that other PCNA-interacting proteins exhibit recruitment kinetics
similar to DNA Ligase I, indicating that PCNA not only serves as a
central loading platform during DNA replication, but also
coordinates the recruitment of multiple enzyme activities to DNA
repair sites. Accordingly, we found that the maintenance
methyltransferase DNMT1, which is known to associate with
replication sites through binding to PCNA, is likewise recruited to
DNA repair sites by PCNA. We propose that DNMT1, like in DNA
replication, preserves methylation patterns in the newly
synthesized DNA, thus contributing to the restoration of epigenetic
information in DNA repair. In summary, we found immediate and
transient binding of repair factors involved in DNA damage
detection and signalling, while repair factors involved in the
later steps of DNA repair, like damage processing, DNA ligation and
restoration of epigenetic information, showed a slow and persistent
accumulation at DNA damage sites. We conclude that DNA repair is
not mediated by binding of a preassembled repair machinery, but
rather coordinated by the sequential recruitment of specific repair
factors to DNA damage sites.