SM10 Flashcards
(23 cards)
What direction does the polymerase read the template strand/parent strand?
three prime to five prime. Strand is synthesized 5 to 3.
What does it mean to say the origin is loaded?
It means the pre-replication complex has formed. (during G1)
What strand has ssbinding proteins?
The lagging strand.
Name the different DNA polymerases.
Alpha - priming (RNA primer) on leading and lagging strand, no 3-5 exonuclease activity
Beta - DNA repair only
Gamma - metochondrial
Delta - primarily lagging strand synthesis, has 3-5 exo
Epsilon- primarily leading strand synthesis, has 3-5 exo
Errors in removal of Okazaki fragments can cause…
insertions or deletions into the DNA
Difference between topoisomerase I vs II.
Type I DNA topoisomerases reversibly cut one strand, the intact strand is passed through the break and the topoisomerase relegates the cut strand relieving the supercoiling. Relieve negative supercoiling in prokaryotes, and both positive and negative supercoiling in eukaryotes. Type I topoisomerase do not use ATP, but instead use the energy from cutting the phosphodiester DNA backbone.
Type II DNA topoisomerases make transient breaks in both strands. The enzyme then passes a second stretch of the double helix through the break and religates the break. This class uses ATP as an energy source. Replication of chromosomes results in interlocked molecules of DNA (precatanenes) and Type II topoisomerases are used to separate them in both prokaryotes and eukaryotes.
What is a precatenane?
When two helices become linked together.
What is DNA gyrase?
Because bacteria have circular chromosomes, DNA gyrase is used to introduce negative supercoils into replicated DNA to relieve the excess positive supercoiling from replication. DNA gyrase is targeted by the class of antimicrobial agents called quinolones, such as ciprofloxacin.
What is role of telomerase in germ, cancer and stem cells?
The telomerase complex contains a protein with reverse transcriptase activity (RNA-dependent DNA polymerase) and a small RNA that acts as a template. The RNA template base pairs with the single-stranded DNA segment of the telomere DNA (on template) and the reverse transcriptase uses it as a template for 5’ to 3’ DNA polymerization, extending the already longer 3’-end of the chromosome (adding additional telomere repeats to template strand). Telomerase then moves to the new end and repeats the process. Once the new strand is sufficiently long, DNA polymerase will bind to 3’end of now longer 3’ template, RNA primer is synthesized and the daughter strand is extended by DNA polymerase as in normal replication.
What does depurination/depyrimidation do?
Take base, leave sugar-backbone intact.
What is deamination of cytosine produce?
Forms uracil.
What are steps of single strand damage repair?
identify, remove, replace, ligate.
Ex: Base excision repair
Nucleotide excision repair
Mismatch repair
What are steps of double-strand break repair?
Caused by ionizing radiation or collapsed replication forks.
Steps: identify, rejoin, ligate
Ex:
Non-homologus end joining (G1, error prone)
Microhomology mediated end joining (sim to NHEJ, S phase, error prone)
Homology directed repair - repair during and after DNA replication
How does BER work?
From damage to single base by oxidation, alkylation (methyl groups), hydrolysis, deamination of adenine to hypoxanthine or guanine to xanthine, or misincorporation of uridine.
Steps:
Removal of the damaged base by a DNA glycosylase forming an apurinic/apyrimidinic or AP site. A single strand break is then made in the DNA strand by AP endonuclease and deoxyribose phosphate lyase remove the single base-free sugar phosphate. DNA polymerase and DNA ligase then fill and seal the nick.
How does NER work?
From damage from helix-distorting lesions such as thymine dimers and 6,4 photoproducts.
Multi-protein complexes recognize the lesion and direct an endonuclease to the site which will cut the damaged strand on each side of the lesion creating a gap. The gap is filled in by DNA polymerases δ and ε and the strand is religated by DNA ligase.
Defect - causes xeroderma pigmentosum
What is NHEJ?
In NHEJ, nuclease chews back both strands. The broken ends are recognized by the repair machinery, brought together and rejoined. The process of preparing the ends for ligation typically involves deletion of stretch of DNA. If a large number of breaks are present, translocations and transversions of chromosomes can occur.
How does HR work?
Homologous repair or homology-directed repair (HR) requires that the cell has mostly completed DNA replication (only active during S and G2 phase). Exchange of strands on sister chromatids and copying of the “good” strand to repair the break ensures a high fidelity of repair.
What is transcription-coupled repair?
If the lesions occur in actively transcribed genes, the RNA polymerase stalls and signals the repair machinery (including BRCA1 and BRCA 2) which will dislocate the polymerase, recruit NER machinery and repair.
How and when does mismatch repair work?
Assoc with replication complex are members of mut family of proteins. They follow replicaiton complex. If they detect site of misincorporation they cause the polymerase to stall. Recruit machinery. Cause cut on either side. Helicase removes this stretch. ssBP stabilizes. Rep complex reforms at site where the DNA segment was removed and reinitiates replication. It is similar to NER but triggered at replication by MUt proteins.
How is DNA damage sensed?
DNA damage sensing is regulated by a pair of protein kinases ATM (ataxia telangiectasia mutated) and ATR (ataxia telangiectasia related) which activate a cascade of events to alter chromatin structure near the damage site and recruit the repair machinery (Brac1-Assoc genome surveillance complex ie BASC). A core complex of DNA repair factors called the Genome Surveillance Complex coordinates the different repair pathways. BRCA1 is the scaffolding protein assoc with this and recruits other proteins in repair process. Otherwise will trigger p53 and pRb for apoptosis/cell arrest
What is Bloom syndrome?
pt have stunted growth, exhibit UV sensitivity and d/t defected DNA helicase.
What is Werner syndrome?
causes premature aging. The Werner gene encodes a DNA helicase.
What is hereditary nonpolyposis colorectal CA?
AD condition that results from mutations in genes involved in DNA mismatch repair.
