Replication Flashcards
Replication
Process of DNA synthesis, occurs during S phase and uses DNA polymerase
Protein + nuclear DNA=
chromatin
Heterochromatin
Very condensed
Contains very few genes. Late replicating and genetically inactive
DNA polymerase needs what to start replicating?
Short stretch of RNA serves as a primer
Topoisomerase
Relieves overwound supercoils (called DNA gyrase in bacteria-inhibited by the quinolone family of antibiotics)
Breaks a phosphodiester bond
Single-stranded DNA binding protein
Binds the single-stranded DNA that has been separated and keeps it exposed in a single-stranded conformation
DNA polymerase α (in complex with primase)
Synthesizes RNA-DNA primer
Primase synthesizes an RNA primer that is 7-10 ribonucleotides long and is extended with 25 nucleotides of DNA by DNA polymerase α. The resulting RNA-DNA primer is complementary to a segment of the parental strand
Euchromatin
Less condensed
DNA and Histone interaction
- 142 hydrogen bonds formed in each nucleosome
- Hydrophobic interactions
- Salt linkages (lysine/argenine are positive and they neutralize negative charge on DNA)
- Histones are highly conserved
Nucleosome
Each nucleosome core particle consists of a complex of 8 histone proteins
DNA polymerase requires what to begin replication?
Primer with a free 3’ OH to begin
Sliding clamp
Keeps DNA polymerase on DNA when moving; releases when double stranded DNA is encountered
Telomerase
Replicates chromosome ends
Special sequence of GGGTTA at end of chromosome
Replenishes these sequences by elongating parental strand in 5’–>3’ direction using an RNA template on the enzyme. DNA polymerase comes in and extends
T-loops: structures protect ends and distinguishes them from broken ones that need to be repaired
Telomere
Telomeres are the caps at the end of each strand of DNA that protect our chromosomes, like the plastic tips at the end of shoelaces. Without the coating, shoelaces become frayed until they can no longer do their job, just as without telomeres, DNA strands become damaged and our cells can’t do their job.
Stem cells and telomeres
Stem cells retain full telomerase activity
Replicative senescence
After many generations, daughter cells will have defective chromosomes and stop dividing; in this way the cell’s lifetime is regulated to guard against cancer
Human fibroblasts (synthesizes the extracellular matrix and collagen) normally divide 60 times before undergoing replicating senescence
Dyskeratosis congenita disease
Carry a mutant telomerase RNA gene
- Premature shortening of telomeres
- Die of progressive bone marrow failure
We need high fidelity replication rates
Germ cells need it to maintain the species
Somatic cells need it to avoid uncontrolled proliferation/cancer
What are the two types of spontaneous DNA damage?
Depurination and Deamination
Depurination
The purine base (adenine or guanine) is removed from the nucleotide via hydrolysis of the N-glycosidic bond between the base and the deoxyribose group. This generates an apurinic (AP) or abasic site in the DNA strand
hydrolysis of the N-glycosyl linkage
Repaired beginning with AP endonuclease
Deamination
The amino group (NH2) of purine or pyrimidine base is hydrolyzed such that adenine is converted to hypoxanthine, guanine is converted to xanthine, and cytosine is converted to uracil, which forms an unnatural deoxyuridine (NH2–> =O)
C–>U
Hydrolysis of the N-glycosyl linkage
Methylated cytosines are problematic
- Occurs at some CpG sequences
- Associated with inactive genes
- Deamination of methyl-C produces T mismatched with G (NH2–> =O)
- A special DNA glycosylase recognizes and removes the T
- Repair is relatively ineffective. Only 3% of C nucleotides are methylated but they account for 1/3 of all point mutations associated with inherited human diseases
What does Direct repair (enzymatic repair) fix?
Pyrimidine/thymine dimers via DNA photolyase
O6 methylguanine via Methylguanine methyltransferase
What does Base excision repair (BER) fix?
Single-base mismatches, nondistorting alterations (i.e. depurination)
How?
DNA glycolases (each recognizes a specific type of altered base), AP endonuclease (cut phosphodiester backbone, damage is removed and gap is repaired), AP lyase (of DNA polymerase β), DNA polymerase β, DNA ligase
