GeneRegulation Flashcards
How is DNA replicated? What complexes are required for replication to proceed?
DNA replication is semi-conservative, and semi-discontinuous. You have the strands split and each strand has two daughter strands. One is part of the parent and one is newly formed. Important complexes include helicase (unwinds DNA), TopI/TopII(supercoiling), RPA (sand binding), primase(primer), RFC(load PCNA), PCNA (sliding clamp), Polymerase(5’-3’ synthesis), RNAseH, FEN1 (remove RNA primer), Ligase(ligate fragments)
Replication is bi-directional
How is replication initiated?
ORC nucleates the stepwise assembly of a pre-replication complex at an ARS and recognizes origin. Must be preset for replication to occur
How are origins recognized(ORC)?
1) In situ detection using radioactive nucleotide incorporation to look for bidirectional origins
2) Molecular mapping to find initiation sites. For example, isolate nascent strands followed by PCR
3) Genetic mapping uses deletion mapping of requires sequences at a few loci
4) Genomic mapping using microarrays and probing genomic DNA from early S phase and looking for a 2-fold increase in signal near (this is the origin)
5) Sequencing methods including Okazaki(OK)-Seq and Repli-seq
How is DNA replication regulated (so that it occurs once and only once)?
Regulated within the cell cycle
phosphorylation of ORC during S phase and this labels it for degradation
What are the obstacles in replication?
1) difficult to replicate regions bc of sequences
2) Structures like G-quadroplexes
3) nucleosomes
4) DNA damage
What are some sources of DNA damage?
Rare( Cosmic radiation, environmental mutagens)
Common( depurination, depryramidinization, cytosine admonition, hydroxyl radicals from mitchondiral respiration, UV irradiation, program breaks due to immune cell development)
How do cells determine what type of repair to use?
- Base hydrolysis = Base excision repair
- Oxidative damage = Base excision repair
- mismatches = mismatch repair
- UV = nucleotide excision repair
- Double stranded breaks = HR & NHEJ
Cell cycle check points –> When and why
- G2/M Phase DSB checkpoint pathway where ATM activated early, and ATR is activated later
p53, CHK1, CHK2 are activated
How do the cell cycle checkpoints and DNA repair pathways interact?
DNA damage repair will shut the cell cycle down if things aren’t going smoothly, and this is regulated by the checkpoints.
How can you measure DNA damage?
H2AX
What are the three types of repair for double stranded breaks and when do they occur?
Homologous recombination( sister chromatid, S and G2 phase), Non-homologous end joining (can be mutagenic, G1 and G2 phase)and single strand annealing(mutagenic, G1 and G2 phase)
Telomere capping prevents…
- Exonucleolytic degration
- activation of DNA damage checkpoint response pathways
- chromosome recombination
- end to end fusions
- homologous recombination
What is a telomere cap?
Caused by a 3’ overhang that creates a t-loop and d loop and its similar to a holiday junction
What is the Telomere protein complex?
Sheltering complexes TRF1/2 bind to sDNA of telomere, POT1, and Ctc1/Stn1/Ten1
Different shelterin components inhibits different DNA damage response pathways
What is Telomerase?
lengthens telomeres
relative of reverse transcriptase
catalytic protein carries a dedicated RNA template
recognizes repetitive sequence of telomeres
