DNA Replication/ Cell Cycle Flashcards
Important points of DNA-protein interactions: typ of bonding location what structures are fitting together requirement
- Hydrogen bonding between amino acid side chain and the nitrogenous bases and backbone is crucial
- most contacts are in the DNA major groove
- alpha helices of the protein associate with the major groove of B-form DNA (site specification)
- Multiple DNA binding domains necessary for site specificity
origins of replication
where DNA synthesis begins, the mammal genome has many of these
Simian virus 40 (SV40):
use in research
why
outcome of research
- model system for DNA replication in humans
- with the exception of t-ag, the saem proteins are used to synth SV40 and human DNA
- identification of many of the proteins involved in human DNA replication
T-ag
- helicase encoded by SV40
- these is a specific binding site for this protein at SV40’s origin of replication
Bloom’s syndrome
high risk of developing malignancies. caused by a deficinency in the helicase protein
Werner’s Syndrome
defective helicase causes premature aging
helicase
seperate the strands of duplex DNA during replication
topoisomerases during DNA replication
undo the supercoiling during replication
Unwinding:
importance
- exposes the bases in the origin of replication
- additional enzymes gain access to the exposed bases in order to copy the strand
DNA primase
synthesizes an approx 10nt RNA primer. this is necessary to innitiate DNA synth
DNA polymerase:
job
reaction type
bond formed
adds nucleotides to the 3’ carbon of the growing chain, synthesizing the chain in the 5’ to 3’ direction. This process is achieved by catalyzing the nucleophilic attack of the preceeding 3’-OH of deoxyribose. a phosphodiester bond is then formed and a pyrophosphate released
Polymerase epsilon
forms the leading strand of newly synthesized DNA in the 5’-3’ direction. This is a high fidelity polymerase in that it has 3’ to 5’ exonuclease activity.
Proliferating Cell Nuclear Antigen PCNA
clamps the polymerases onto the DNA template
Replication factor C
helps load polimerase delta and epsilon onto the DNA
overall direction of growth of the lagging strand
3’ to 5’ although the individual okazaki fragments are synthesized 5’ to 3’
pol alpha primase
plays a key role in the synthesis of okazaki fragments since a new RNA primer must be laid down before the synthesis of each okazaki fragment. It is also required to initiate leading strand synthesis.
removal of RNA primer and suturing of okazaki fragments
RNA primers are removed by a 5’ to 3’ exonuclease and the holes filled in by pol delta and finaly bonded to the okazaki fragments by DNA ligase
DNA ligase: function process (mechanism)
seals single stranded nicks in the daughter strand. is activated when ATP donates AMP to form an enzyme-AMP complex. AMP is then transferred to the 5’ phosphate on the nick, activating the phosphate so it can form a covalent linkage with the adjacent 3’-OH
pol gama
responsible for mitochondrial DNA synthesis
pol beta
plays a role in repair events
AZT:
how it works
effectiveness
problems
- nucleotide anologue that inhibits the reverse trancriptase of the HIV.
- effectiveness limited due to toxicity (it is a substrate for pol gama, disrupting mitochondrial DNA synthesis)
acyclovir
nucleotide anologue which inhibits the DNA pol present in the herpes virus
Cell Cycle subdivisions
G1, S, G2, M
can also enter quiscent stage called G0
S phase
-when DNA replication occurs
G1
long period of growth preceeding the replication of DNA.
Begin immediately after mitosis
G2
additional time of growth before entering mitosis
cyclin-dependent kinase
regulate the progression of a cell through the cell cycle. composed of a cylcin and a kinase
p53: function oncological classification
- protein that functions at the G1/S checkpoint and helps to prevent the cel from entering S phase if the cell has damaged DNA. DNA can either be repaired and cell will enter S or the cell will apoptose
- tumor supressor
Li-Fraumeni Syndrome:
cause
effect
- inheritance of a mutated form of p53
- at risk for developing tumors at multiple sites
mismatch repair:
- when
- identifying the incorrect nucleotide
- identifying daughter strand
- communication of associated enzymes
- process after incision
- occurs when DNA is incorrectly replicated
- the mismatch is recognized by an enzyme, Mut S
- templates strands have methylated adenine residues in d(GATC) sites and these sites are recognized by Mut H which upon recognition makes an incision in the strand
- it is thought the Mut L acts as a protein-protein interface between Mut H and Mut S
- error containing strand removed by a helicase and exonuclease and the empty space is refilled by a DNA pol, an SSB and DNA ligase
trinucleotide repeat repair
- thought to be done via the mismatch repair pathway
- Thought to may have something to do with the high number of trinucleotide repeats associated with the huntingtons disease mutation
excision repair:
function
steps
detail of each step (proteins involved)
- removal of a faulty DNA segment and its replacement by DNA synthesis
- steps in E.coli: incision, excision, resynthesis, ligation
- Incision: the uvrABC complex cleaves the damaged DNAon either side of the lesion creating a small fragment
- excision: fragment is removed from the complementary strand, leaving a small gap
- resynthesis: gap is filled by DNA polymerase 1 using the 3’-OH end of the nicked strand as a primer. DNA ligase then joins the strands
xeroderma pigmentosum
- results from biochemical defects in the excision repair system
- afflicted generally die from metastases of malignant skin tumors before 30
- examples of the importance of the DNA repair process
removal of uracil from DNA:
why necessary
- cytosine spontaneously deaminates to form uracil. this causes a mutation in the gene
- this uracil is recognised by uracil-DNA glycosidase which hydrolyzes the glycosidic bond between uracil and deoxyribose
- this formas an AP (apurinic/apyrimidinic) site which is recognized by an AP endonuclease which removes the nitrogenous base-free backbone, initiating an excisionr repair-like process
substitutions, deletions, and insertions
substitutions: swapping of a base pair
deletion: deletion of a base pair
insertion: insertion of a base pair
chemical mutagens
- environmental factors that produce genetic mutations
- many are substances that react with DNA and cause modifications of bases. some cause changes in base pairing specificity
deamination
nitrous acid reacts with bases that contain amino groups and can convert cytosine and adenine into uracil and hypoxanthine, respectively
alkylation
- major site for this reaction is the N7 position on guanine
- methylation at this postion cause labilization of the N-glycosidic linkage in DNA and leads to the loss of guanine residues from DNA
Polycyclic hydrocarbons:
- what and how
- examples
- mutation type
- have the capacity to intercalate between adjacent basse pairs in DNA leading to insertion or deletion of one or more base pairs
- proflavin and ethidium bromide
- result in frameshift muations
radiation
causes the removal of electrons from molecules causing alterations in the gene structure
pol alpha primase pol beta pol delta pol epsilon pol gama
- pol alpha primase: laying down the RNA primers for leading strand and okazaki fragments
- beta: plays a role in repair events
- delta:required for lagging strand synthesis
- epsilon: required for leading strand synthesis
- gama: replicates mitochondrial DNA
