In spite of accumulating evidence suggesting the important role of the TS pathway for genomic stability and possibly for suppression of carcinogenesis, the detailed molecular mechanisms of the TS pathway and proteins participating in this pathway are not clearly understood even in yeast. The requirement of the Rad52 protein but not the Rad51 protein in the TS pathway (21
) suggests that the TS pathway might not require Rad51-coated single-stranded DNA as an intermediate structure (). One intriguing model called “fork reversion” could explain the ability to bypass DNA damage without using Rad51 and strand invasion (). In this model, the stalled replication forks would be reversed until the stalled replicating strand and its replicated sister strand could be hybridized. This fork reversion would allow the DNA polymerase to use the newly replicated sister strand as a template to extend the stalled replicating strand. When the DNA replication machinery reached the end of the sister strand, an X-shaped structure would then be resolved and the replication fork would be reestablished. Because the shape generated during fork reversion resembles that of a chicken foot, the fork reversion model is also known as the chicken foot model. Although the fork reversion model seems to be a very attractive molecular mechanism for the TS pathway, there is no evidence that can exclude that the template is simply switched without fork reversion to initiate the pathway. Alternatively, stalled forks are simply marked by the PCNA modification, and another replication origin in eukaryotes could be fired to continue DNA replication (22
Figure 2 The hypothetical molecular model of the TS pathway includes DNA replication fork reversion to create a chicken foot-like DNA structure. Several important steps are described in the clockwise direction. X and Y represent unknown interacting proteins such (more ...)
We characterized the initiation of the TS pathway through the PCNA polyubiquitination by Rad5/SHPRH; however, it is still unclear what the next step in this process could be. K63-mediated PCNA polyubiquitination has not been linked to the proteosomal degradation of PCNA and therefore appears to be a signal for pathway switching. If this is the case, there might be specific proteins recruited to polyubiquitinated PCNA. The discovery of proteins interacting with polyubiquitinated PCNA will help clarify these downstream events.
What could then facilitate the next step of fork reversion, which is proposed to be a change in DNA structure? Yeast Rad5 and SHPRH both have Swi2/Snf2 ATPase–helicase domains, which could promote the modification of DNA (). Therefore, besides its role for PCNA polyubiquitination, SHPRH could also facilitate the modification of DNA structures or the remodeling of chromatin structure for fork reversion. Proteins specifically recruited through their interaction with polyubiquitinated PCNA could directly or indirectly participate in this step.
Figure 3 Yeast Rad5 and human SHPRH share similar domain structures. Both proteins have a RING domain responsible for proliferating cell nuclear antigen (PCNA) polyubiquitination and seven Swi2/Snf2 ATPase/helicase domains. NLS, LH, and PHD stand for nuclear localization (more ...)
When the DNA replication fork has been reversed and the sister strand has been annealed to the reversed strand, the replicative DNA polymerase could then function in normal DNA replication mode (). However, when DNA replication approached the end of the sister strand, the chicken foot–like DNA structure would have to be resolved. The resolution of this structure could occur through simple branch migration or perhaps a helicase could facilitate this event.
Polyubiquitinated PCNA at the reestablished DNA replication fork should be removed for normal DNA replication. This could be achieved by the degradation of the ubiquitin chain by a deubiquitinating (DUB) enzyme. A mammalian DUB enzyme, USP1, was discovered and found to deubiquitinate monoubuiquitin from PCNA (23
). Recently, RAP80 was identified as a DUB enzyme to deubiquitinate K63-linked polyubiquitin chain (24
). It is possible that USP1 or RAP80 functions to remove the polyubiquitin chain from PCNA after the reestablishment of the DNA replication fork.
Lastly, it is still unclear whether the TLS and TS pathways are equally or specifically used in different situations. In yeast studies, the inactivation of the TS pathway by the rad5
mutation enhanced TLS-dependent mutagenesis (25
). Therefore, at least in yeast, both pathways are somewhat in competition. It is still unclear what could trigger the activation of the different pathways. Although thorough studies are necessary, it could be possible that different DNA adducts, different DNA sequences, different phases of the cell cycle, or leading vs lagging strands could affect this choice.
Emerging studies have begun to uncover the importance of the RAD5-dependent TS pathway for the suppression of genomic instability and its putative role for the suppression of carcinogenesis. Further studies that will answer many questions posed in this review and new animal models that can demonstrate its direct role in tumorigenesis will be beneficiary for understanding at least a specific role of DNA damage bypass for the suppression of cancer development.