DNA Replication and Repair Flashcards
semi-conservative replication
single helix separated into 2 strands, each serving as a template for replcation of a new complementary strand
each daughter molecule contains one strand from the parent helix and one new strand
prepriming complex
a complex of proteins that binds to the origin of replication and begins to unwind the helix for replication
Describe the initiation of DNA replication in both prokaryotes and eukaryotes.
very similar between prokaryotes and eukaryotes
DNA helicase
after generation of hte replication bubble, helicase binds and starts to unwind DNA, forcing the two strands apart
ATP is required as an energy source
once the strands are opened, binding proteins attach to prevent reannealing
MCM helicase
protein that binds the ORC and initiates formation of the replication bubble, only happens once per cell cycle
regulatory mechanism to ensure correct amounts of DNA are produced
separates the two strands during replication as it moves down the helix
topoisomerases
two types, TOPO1 and TOPO2 to deal with supercoils generated by the unwinding of DNA
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
relieves both positive and negative supercoiling in eukaryotes, do not use ATP
type II DNA topoisomerases
make transient breaks in both strands, then passes a second stretch of the double helix through the break and religates the break
uses ATP as an energy source
called DNA gyrase in bacteria
replication fork
leading strand is synthesized 5’ to 3’
lagging strand is also snythesized 5’ to 3’ but in fragments called Okazaki fragments
primase
an RNA polymerase that synthesizes a short ~10 nucleotide long stretch of RNA that is complementary to the parent strand
addition of primase to the prepriming complex creates the primosome
DNA Pol alpha
responsible for synthesizing RNA primeras on the lagging strand as the replication fork moves
DnaG in prokaryotes
DNA polymerase epsilon
elongates the replicating DNA in the leading strand in eukaryotic cells
done by DNA polymerase II in prokaryotes
polymerase delta
elongates the DNA strande in the lagging strand in eukaryotic cells
done by DNA polymerase II in prokaryotes
PCNA
proliferating cell nuclear antigen - pentamer through which the DNA passes, acts as a sliding clamp around the DNA
binds polymerase and prevents it from falling off
also bound to helicase
Flap Endonuclease I (FENI)
removes the RNA and DNA flap after it the RNA is displaced by pol delta
after cleavage, DNA ligase comes and seals the gap
proofreading
DNA polymerase has 3’ to 5’ exonuclease activity so mispaired bases can be removed and re-polymerized
pol alpha does not have proofreading capability so errors in the Okazaki fragment are removed during its removal
FACT
helps remove histones from the replication fork and then adds them back in
histone octamers stay intact when they are removed
new histones are made during the S phase when DNA is replicating
telomerase
if telomeres shorten sufficiently beyond a critical length, the cell activates a pathway to permanently stop dividing
telomerase conatains a protein with reverse transcriptase activity and a small RNA that acts as a template
retrotransposons
transposons that utilize RNA intermediates
types of single-strande damage repair
base excision repair, nucleotide excision repair, mismatch repair
types of double-strand break repair
non-homologous end joining
microhomology-mediated end joining
homologous recombination (homology directed repair)
sources of DNA damage
errors in insertion, wrong base, insertion of extra molecules, environmental damage such as radiation or oxidization
base excision repair (BER)
removes non-helix distorting lesions, most common form of DNA damage and involves the modification of bases by oxidation, alkylation, deamination, or misincorporation
short pathc BER, gap filled by DNA polmerase
long patch BER, a new DNA strand is laid down by the machinery and the old flap is excised by a FLAP endonuclease
mismatch repair
fixes errors caused by misincorporation of bases during DNA replication, reocgnizes parental strand due to methylation in prokaryotes and maybe by nicks in eukaryotes
nucleotide excision repair
removes helix distorting changes such as pyrimidine dimers and oxidized benzo[a]pyrene
endonucleases cut damaged strand on each side of the lesion, gap filled by DNA polymerase delta and epsilon, strand religated by DNA ligase
transcription coupled repair
if RNA polymerase detects lesions in DNA, wills tall and signal for repair components, defects in XP proteins in this process result in xeroderma pigmentosum
non-homologous end joining (NHEJ)
broken ends are recognized by the repair machinery, brought together and rejoined
process of preparing ends for ligation typically loses some of the DNA resulting in mutations
homologous repair or homology directed repair (HR)
utilizes the machinery used for recombination between chromosomes during meiosis, requires that the cell has mostly completed DNA replication
H2AX
histone that can detect breaks in the DNA, will spread down chromatin to indicate to the machinery that there is an error in this region
genome surveillance complex
cooridnated by BRCA1
ATM and ATR are sensing kinases that phosphrylate BRCA1 an activate the complex
BLM - bloom syndrom protein, helicase involved in double stranded break repairs
chromosome translocations
reciprocal translocation - exchange of two fragments
robertsonian translocation - exchange in proximal short arms, some DNA is lost
chromothripsis
thousands of clustered chromosomal rearrangements occur in a single event in colcalised and confined genomic regions in one or a few chromosomes
pieces religate together differently, can generate lost segments and boule double minutes (pieces of circular DNA)
xeroderma pigmentosum
AR disease that cuases extreme sensitiviety to light, abnormal skin cancers develop, cells defective at excising thymine dimers caused by UV radiation
Bloom’s syndrome
patients have stunted growth, exhibit UV sensitivity and have defective DNA helicase (BLM)
Werner syndrome
causes “premature aging” effects, such as graying, cataracts, osteoporosis, werner gene encodes a DNA helicase involved in repair
hereditary nonpolyposis colorectal cancer
an AD condition that results from mutations in genes involved in DNA mismatch repair
