DNA Replication and Repair (1B) - Bio & Biochem

1. What is the primary function of single-strand binding proteins (SSB) in replication?

   Stabilize ssDNA and prevent reannealing or secondary structure formation. SSBs coat exposed single strands to maintain their accessibility and prevent unwanted structures during replication.

2. In bacteria, which DNA polymerase primarily removes RNA primers and fills in DNA?

   DNA polymerase I. Pol I uses its 5' to 3' exonuclease to excise RNA primers and polymerase activity to replace them with DNA.

3. What is the function of primase during DNA replication?

   Synthesizes short RNA primers to provide a free $3'$-OH. Primase, an RNA polymerase, creates RNA segments that DNA polymerase extends, as it cannot start chains de novo.

4. What chemical group provides the nucleophile that attacks the incoming dNTP during elongation?

   The primer strand $3'$-OH group. The 3'-OH attacks the alpha phosphate of the incoming dNTP, forming a phosphodiester bond during chain elongation.

5. What is the specific base-pairing rule that stabilizes DNA during replication and repair?

   A pairs with T; G pairs with C (Watson–Crick base pairing). Specific hydrogen bonds between purines and pyrimidines ensure fidelity in template-directed synthesis and repair.

6. What is semiconservative DNA replication?

   Each daughter duplex contains one parental strand and one newly synthesized strand. This mechanism, proven by Meselson-Stahl, preserves genetic information by using each parental strand as a template.

7. What is nucleotide excision repair (NER) primarily used to fix in DNA?

   Bulky, helix-distorting lesions (e.g., thymine dimers) by excising an oligonucleotide. NER excises a short single-stranded segment containing the lesion, then synthesizes a replacement using the intact strand.

8. What is the overall direction of DNA synthesis by DNA polymerases in all organisms?

   DNA is synthesized in the $5' \to 3'$ direction. DNA polymerases add nucleotides to the growing chain's 3' end, ensuring synthesis proceeds from 5' to 3'.

9. What is mismatch repair (MMR) primarily responsible for correcting after replication?

   Base-base mismatches and small insertion/deletion loops that escape proofreading. MMR detects and excises errors like mismatches or loops post-replication, using strand discrimination to guide repair.

10. What is the direction that the DNA template strand is read by DNA polymerase?

    The template is read in the $3' \to 5'$ direction. Antiparallel strand orientation requires the template to be read 3' to 5' for new strand synthesis in the 5' to 3' direction.

11. What is the role of the sliding clamp (e.g., PCNA in eukaryotes) in DNA replication?

    Increases processivity by tethering DNA polymerase to DNA. The clamp encircles DNA and binds polymerase, enhancing its ability to synthesize long stretches without dissociating.

12. What is the primary function of topoisomerases during DNA replication?

    Relieve supercoiling/torsional strain ahead of the replication fork. They introduce temporary strand breaks to relax positive supercoils generated by unwinding at the fork.

13. What is the immediate energy source that drives phosphodiester bond formation in DNA synthesis?

    Cleavage of dNTP to dNMP with release of pyrophosphate ($PP_i$). Hydrolysis of the high-energy bonds in dNTP releases pyrophosphate, providing the energy to make the reaction exergonic.

14. What is the core function of telomerase in eukaryotic cells?

    Extends the $3'$ end of telomeres using an internal RNA template (reverse transcriptase). As a ribonucleoprotein with reverse transcriptase activity, telomerase adds repeats to prevent telomere shortening.

15. What is the key limitation of DNA polymerases that necessitates a primer?

    They cannot initiate synthesis de novo; they require a preexisting $3'$-OH. DNA polymerases can only extend existing chains, requiring an RNA primer to supply the initial 3'-OH group.

16. In bacteria, which DNA polymerase primarily performs chromosomal DNA elongation?

    DNA polymerase III. Pol III is the highly processive replicative enzyme responsible for most nucleotide addition during bacterial replication.

17. What is the replication fork "lagging strand" defined by relative to fork movement?

    The strand synthesized discontinuously as Okazaki fragments. Synthesis occurs in short segments away from the fork due to the antiparallel nature of DNA strands.

18. What is the "end-replication problem" that occurs in linear eukaryotic chromosomes?

    After primer removal, the terminal lagging-strand segment cannot be fully replicated. Lagging strand synthesis leaves a gap at the 5' end after RNA primer removal, as no upstream 3'-OH is available for filling.

19. What is the function of DNA ligase in replication and repair?

    Seals nicks by forming phosphodiester bonds between adjacent DNA ends. It catalyzes the ATP-dependent formation of phosphodiester bonds to join Okazaki fragments or repair breaks.

20. What is the primary function of helicase during DNA replication?

    Unwinds the parental double helix at the replication fork. Helicase uses ATP hydrolysis to separate DNA strands, creating single-stranded templates for polymerase access.

21. What is base excision repair (BER) primarily used to fix in DNA?

    Small, non-bulky base lesions (e.g., deamination, oxidation) via DNA glycosylase. BER initiates with glycosylase removing altered bases, followed by strand incision and gap-filling for subtle damage.

22. What is the key mechanistic difference between topoisomerase I and topoisomerase II?

    Topo I cuts $1$ strand; topo II cuts $2$ strands (ATP-dependent). Type I relaxes supercoils via single-strand nicks without ATP, while type II passes strands through double-strand breaks using ATP.

23. Identify the most accurate classification of UV-induced thymine dimers as DNA damage.

    Bulky, helix-distorting lesions typically repaired by NER. UV light causes covalent bonds between adjacent thymines, distorting the helix and triggering NER for removal.

24. What is the replication fork "leading strand" defined by relative to fork movement?

    The strand synthesized continuously in the same direction as fork movement. It allows continuous elongation toward the replication fork without interruption as the helix unwinds.

25. Which enzymatic activity enables many DNA polymerases to proofread newly added bases?

    $3' \to 5'$ exonuclease activity. This activity removes incorrectly paired nucleotides from the 3' end, allowing replacement before synthesis continues.

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