DNA Replication (Lec. 10) Flashcards
Chapter 7
Compare the roles of DNA polymerases in E. coli with those in mammalian cells
In E. coli, DNA polymerase I is not the major enzyme responsible for DNA replication. Instead, it’s principally involved in repair of damaged DNA
Contrast the mechanisms of synthesis of the leading and lagging strands of DNA
The leading strand is synthesized continuously in the direction of replication fork movement. The lagging strand is synthesized in small pieces (Okazaki fragments) backward from the overall direction of replication. The Okazaki fragments are then joined by DNA ligase.
Identify the proteins found at replication forks of bacteria and mammalian cells
DNA ligase joins Okazaki fragments. Primase synthesizes primers (short fragments of RNA complementary to the lagging strand). DNA polymerase synthesizes a strand of DNA complementary to the leading strand, and also the Okazaki fragments. RNase H removes the RNA primers. Sliding-clamp proteins load the polymerase onto the primer and maintain its stable association with the template. Clamp-loading proteins load the sliding-clamp proteins onto the DNA. Helicases unwind the DNA. Single-stranded DNA-binding proteins keep the two strands of DNA apart. Topoisomerases catalyze to reversible breakage and rejoining of DNA strands (to prevent DNA from twisting around itself).
Describe the mechanisms that ensure accurate DNA replication
The free energy differences resulting from changes in hydrogen bonding between correctly matched and mismatched bases favor the formation of correctly matched bases. DNA polymerase actively discriminates against incorporation of a mismatched base by adapting to the conformation of a correct base pair. Replicative DNA polymerases operate in the reverse direction of DNA synthesis (3’ to 5’), and participate in proofreading newly synthesized DNA.
Compare origins of replication in bacteria and mammalian cells
Bacterial DNA is circular, so two replication forks form and move in opposite directions along the chromosome, and there’s only a single origin of replication. Eukaryotes have much larger genomes, so multiple origins are necessary, and replication proceeds in both directions along the chromosome
Summarize the action of telomerase
It’s a reverse transcriptase that catalyzes the synthesis of telomeres in the absence of a DNA template (it carries its own template RNA which is complementary to the telomere repeat sequences).
Compare and contrast direct repair of DNA damage with the different types of excision repair
Direct repair (aka photoreactivation) is when energy derived from visible light is utilized to break a cyclobutane ring structure that formed between two adjacent pyrimidine bases after exposure to UV irradiation. Base-excision repair is where single damaged bases are recognized and removed from DNA. Nucleotide-excision repair is where damaged bases are removed as part of an oligonucleotide containing the lesion. Mismatch repair is where mismatched bases specifically from newly replicated DNA strands are excised.
Explain why defects in DNA repair lead to cancer
Several genes are involved in the mismatch repair system, and defects in these genes appear to result in a high frequency of mutations in other cell genes, wit a correspondingly high likelihood that some of these mutations will eventually lead to the development of cancer by affecting genes that regulate cell proliferation.
Describe translesion DNA synthesis
Replication of damaged DNA by specialized polymerases. Provides a mechanism by which the cell can bypass DNA damage at the replication fork, which can then be corrected after replication is complete.
Summarize the mechanisms cells use to repair double-strand breaks
Recombinational repair:
1) simply rejoin the broken ends of a single DNA molecule (issue here is that there’s a high frequency of errors resulting from deletion of bases around the site of damage.
2) homologous recombination with DNA sequences on an undamaged chromosome, which provides a mechanism for repairing damage and restoring normal DNA sequence.
Describe the rearrangements in immunoglobulin heavy and light chains
Class-switch recombination results in the production of antibodies with distinct functional roles in the immune response. Somatic hypermutation increases the diversity of immunoglobulins by producing multiple mutations within rearranged variable regions of both heavy and light chains.
Explain how nonhomologous end joining of double-strand breaks and cytosine deamination contribute to immunoglobulin diversity.
Cytosine deamination introduces U in place of C in DNA. Removal of U by base excision repair leaves a single-strand gap in the DNA
Explain why DNA amplification increases gene expression.
Gene amplification increases the number of copies of a gene within a cell through repeated rounds of DNA replication. In cancer cells, it frequently results in the increased expression of genes that drive cell proliferation.
What is the DNA polymerase family? In what species was it first identified?
It’s a group of enzymes responsible for synthesizing new DNA (from a template) and fixing damaged DNA. It was first found in E. coli
What are the polymerase names in bacteria? In eukaryotes?
Bacteria have polymerase I, II, and III. Eukaryotes have alpha, delta, and epsilon polymerase, and also gamma polymerase for mitochondrial DNA.
What do the different polymerases all have in common?
Can only synthesize DNA in the 5’ to 3’ direction, and can only add nucleotides to a primer (can’t initiate DNA synthesis on its own)
What direction does DNA polymerase READ? In what direction does it BUILD?
Reads 3’ to 5’, builds 5’ to 3’
What is a replication fork?
An active site of DNA synthesis
Can you describe the leading and lagging strands?
yes
What are primers synthesized by? Are they made of DNA or RNA?
Primase (which is an RNA polymerase, because primers are made of RNA)
How many primers will the leading strand have? And the lagging strand?
The leading strand will have one, the lagging strand will have multiple
What do clamp-loading and sliding-clamp proteins do?
help DNA polymerase bind to and remain associated with the DNA template
What do helicases do?
Unwind DNA
What do DNA-binding proteins do?
stabilize unwound DNA strands
What do topoisomerases do?
break and rejoin DNA strands to relieve tension from unwinding
What is a replisome
The whole complex of enzymes working together during DNA replication
What are histone chaperones?
Recall that nucleosomes are histones + DNA. Histone chaperones disassemble nucleosomes to allow for replication, and add new histones to the new strands
What is translesion DNA synthesis?
Replication across a site of DNA damage
What are telomeres?
Non-coding repeating sequences at the end of each chromosome that shorten with each cell division. Cells will stop dividing (senescence) when they run out of telomere.
What is telomerase?
An enzyme that can extend the telomeres. They have high activity in a few cells (e.g. embryonic stem cells), and mutation can lead to cancer development
Why is DNA rearrangement important? When does it happen?
Helps to add diversity to the gene products we can make, and important for the diversity of the vertebrate immune system. Genes rearrange during development of B cells.
What is somatic hypermutation?
Cell creates mutations in the variable region of heavy and light chains , and deamination of cytosine creates uracil. The process of fixing this change is error-prone, leading to mutations that stay in the DNA
What is gene amplification?
Results from repeated rounds of DNA replication at a specific region of the genome; increases the number of copies of genes within a cell and therefore the expression of those genes; can occur normally during development or abnormally in cancer cells.
