DNA Metabolism

What proteins are required for DNA replication and what is the mechanism of lagging strand synthesis?

● DNA replication is semiconservative, with each daughter molecule composed of one newly synthesized strand and one template strand. DNA replication begins at an origin and proceeds in both directions using two replication forks.

● Each replication fork produces one leading strand and one lagging strand. The leading strand is synthesized continuously, while the lagging strand is synthesized discontinuously, producing Okazaki fragments. The presence of multiple β clamps allows for rapid lagging strand synthesis.

● An active prokaryotic replication fork requires topoisomerase, DNA helicase, primase, single-stranded DNA binding proteins, the core polymerase, and a spare β clamp for synthesis of the next Okazaki fragment. Binding of helicase is a critical part of the initiation process.

● High fidelity is needed to eliminate errors of replication. DNA polymerases discriminate by proper base pairing, because base pairs that are not Watson–Crick base pairs do not fit the geometry of the active site. Improperly inserted bases can be removed by the proofreading function of polymerases.

● Prokaryotic replication has one origin and one site of termination opposite the origin. Eukaryotic replication involves multiple origins on each chromosome, and origins are activated at different times in the cell cycle. Because of their linear nature, eukaryotic chromosomes have telomeres at the chromosome ends that shorten with each replication process unless the enzyme telomerase is present.

How is DNA damaged and what proteins are required for DNA repair?

● DNA damage can lead to mutations, depending on the severity of the damage. Common damaging agents include reactive oxygen species, UV light, alkylating agents, and errors of replication.

● Mutations to a single nucleotide can be silent or can cause changes to gene products through either single amino acid substitutions or production of a truncated protein. Mutations can lead to a variety of issues, including cancerous cell growth. Generally, many mutations must accumulate in a cell before it becomes cancerous.

● Cells have the ability to repair DNA, including mismatch repair, base excision repair, and nucleotide excision repair. Single-strand DNA breaks and double-strand DNA breaks also must be repaired.

● Some DNA repair occurs by highly specialized systems that deal with only one type of damage. Photolyase specifically reverts cyclobutane pyrimidine dimers to the original pyrimidines. O6-methylguanine-DNA methyltransferase (MGMT) is the only mechanism for removal of O6-methylguanine, a common alkylated form of guanine.

What biochemical processes are required for DNA recombination?

● Homologous recombination requires a double-strand break to allow the formation of a Holliday junction. Resolving the Holliday junction produces either a crossover product or reversion to the original chromosomes.

● Integration of a viral genome into host DNA is a recombination process involving a double-strand break, mediated by proteins from both the virus and the host cell. Bacteriophage λ integrates its genome into a specific site on the E. coli chromosome, allowing for passive replication of the viral genome each time the E. coli chromosome is replicated. Alternatively, lysis of the cell can be initiated by production of a large number of phage virions.

● HIV uses reverse transcriptase to convert its single-stranded RNA genome into double-stranded DNA prior to insertion into the host genome. The viral integrase enzyme is required for integration, along with several host proteins associated with double-strand break repair.

● Recombination of immunoglobulin genes has the capacity to generate billions of different antibodies through the process of V(D)J recombination and junctional diversification.

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