7.5
Translesion DNA Polymerases
Translesion (TLS) polymerases rescue stalled DNA polymerases at sites of damaged bases by replacing the replicative polymerase and installing a nucleo…
During replication, a sliding clamp, which is a β-subunit in bacteria or proliferating cell nuclear antigen - PCNA - in eukaryotes, fastens the polymerase to the DNA as it moves along the strand, catalyzing the new DNA synthesis.
When this replicative polymerase gets stalled on a damaged base or region, specialized enzymes covalently modify this sliding clamp through the addition of ubiquitin or SUMO proteins. The modification triggers the release of the replicative polymerase and recruitment of a special polymerase called Translesion DNA polymerase, or TLS polymerase, to the damaged site through interactions with the clamp.
Next, the TLS polymerase inserts a nucleotide across the damaged site in a process called “Translesion DNA synthesis”. Once the nascent DNA chain has extended beyond the lesion, the chemical modification is detached from the clamp, and the TLS polymerase is switched with the cell’s replicative DNA polymerase. With the binding of the replicative polymerase, accurate DNA replication resumes.
Unlike damage repair, there is no restoration to the original DNA sequence, and the damage or lesion will still be present in the DNA, so the phenomenon is described as damage tolerance.
During replication, while some TLS polymerases can add the correct nucleotides to the new strand by chance, others may be prone to errors that give rise to mutations. The type of lesion often determines the accuracy of the polymerase. For example - where the error is an abasic site, there is no coding information for the polymerase to add the correct nucleotide during replication. In archaea, the polymerase Dpo4 preferentially adds an Adenine opposite an abasic site - but other polymerases may fix similar lesions in different ways.
Q1: What happens when a replicative polymerase encounters DNA damage during replication?
When a replicative polymerase stalls on a damaged base, specialized enzymes modify the sliding clamp by adding ubiquitin or SUMO proteins. This modification triggers release of the replicative polymerase and recruits a translesion DNA polymerase to the damaged site. The TLS polymerase then inserts a nucleotide across the lesion, allowing replication to continue.
Q2: How do translesion DNA polymerases differ structurally from regular replicative polymerases?
Y family translesion polymerases lack the 3' to 5' exonuclease proofreading domain found in replicative polymerases. They possess a larger, more open active site that accommodates bulky, chemically modified bases, including covalently linked bases in thymine-thymine dimers. These structural features enable TLS polymerases to bypass DNA lesions that would stall regular polymerases.
Q3: What is damage tolerance and how does it differ from DNA repair?
Damage tolerance allows cells to continue DNA replication past lesions without restoring the original DNA sequence. Unlike repair mechanisms, the damaged base or lesion remains in the DNA after translesion synthesis. This process buys time for the cell to repair damage through other pathways while maintaining replication continuity.
Q4: Why must translesion polymerases extend the DNA strand beyond the lesion before switching back to replicative polymerase?
If the replicative polymerase resumes immediately after nucleotide insertion, its 3' to 5' exonuclease proofreading activity will recognize and remove the inserted base. By extending several nucleotides beyond the lesion, the TLS polymerase positions the inserted base away from the active site, protecting it from removal when the replicative polymerase takes over.
Q5: How does the type of DNA lesion affect translesion polymerase accuracy?
Different lesions present different coding challenges. For abasic sites, which lack coding information, polymerases must insert nucleotides without template guidance. The archaeal polymerase Dpo4 preferentially adds adenine opposite abasic sites, while other polymerases may handle similar lesions differently. Some TLS polymerases insert correct nucleotides by chance, while others are error-prone and generate mutations.
Q6: What role do ubiquitin and SUMO modifications play in translesion DNA synthesis?
Ubiquitin and SUMO proteins are covalently added to the sliding clamp when replicative polymerase stalls on damaged DNA. These chemical modifications signal the release of the stalled polymerase and recruit translesion DNA polymerase to the lesion site. Once the TLS polymerase extends the strand beyond the damage, the modification is removed from the clamp, allowing replicative polymerase to resume.
Q7: How does translesion DNA synthesis relate to restarting stalled replication forks?
Translesion DNA synthesis allows cells to bypass lesions and continue replication when forks stall at damaged bases. By inserting nucleotides across lesions and extending the strand, TLS polymerases enable replication to proceed past obstacles that would otherwise halt fork progression. This mechanism works alongside other cellular responses to restarting stalled replication forks.