DNA Replication Flashcards
Is the process by which a double-stranded DNA molecule is copied to produce two identical DNA molecules.
DNA Replication
- Replication of DNA
–base pairing allows each strand to serve as a template for a new strand
–new strand is 1/2 parent template & 1/2 new DNA
Semi-conservative
is an unwound and open region of a DNA helix where DNA replication occurs.
Replication bubble
Which enzyme unwinds DNA
Helicase
What stabilizes the DNA helix unwound by helicase
single-stranded binding proteins
Steps of DNA Replication
- Unwind DNA
- Build daughter DNA strand
is an enzyme that synthesizes short RNA sequences called primers.
DNA primase
Enzyme that relieves additional coiling during DNA replication
Topoisomerase
How long are RNA primers
5-10 nucleotides long
nick sealing by _________ joins new Okazaki fragment to the growing chain
“spot welder” enzyme
DNA ligase
serves as starter sequence for DNA polymerase III
RNA primer
removes sections of RNA primer and replaces with DNA nucleotides
DNA Polymerase I
Loss of bases at 5’ ends in every replication
- chromosomes get shorter with each replication
Chromosome erosion
telomere sequence in human
TTAGGG
- enzyme that extends telomeres
- can add DNA bases at 5’ end
- different level of activity in different cells
Telomerases
Is the enzyme responsible for maintenance of the length of telomeres by addition of guanine-rich repetitive sequences
Telomerase
the number of times a normal human cell can divide before it stops dividing and enters a state of cellular senescence
Hayflick limit
Holds DNA polymerase in place
Sliding Clamp
Prevents the DNA double helix from getting too tightly wound as the DNA is opened up
Topoisomerase
A method for amplifying DNA segments using cycles of denaturation, annealing to primers, and DNA polymerase-directed DNA synthesis
Polymerase Chain Reaction (PCR)
3 Steps of performing PCR
- Denaturation at 95-96°C
- Annealing at 55-68°C
- Elongation (Extension) at 72°C
PCR Components:
- DNA sample
- Primers
- Nucleotides
- Taq polymerase
- Mix buffer
- PCR tube
Machine used for PCR
Thermal cycler
Step in PCR:
DNA is heated to break the hydrogen bonds between the two polynucleotide strands
–Two single-stranded DNA molecules serve as templates
Denaturation
Step in PCR:
Short nucleotide sequences (primers for DNA replication) are mixed with the DNA and bind to complementary regions on single-stranded DNA
–Takes place at lower temperature
–Primers are 20-30 nucleotides long, synthesized in the laboratory
Annealing
Step in PCR:
The enzyme Taq polymerase is added to synthesize a complementary DNA strand
–Taq is a DNA polymerase from a bacterium found in hot springs
DNA Synthesis
Process wherein a cell recognize and rectify damage to the DNA molecules
DNA Repair Mechanism
Sources of DNA damage
– Endogenous (ROS, replication errors)
– Exogenous (UV, cigarette smokes)
Common causes of DNA damage
Base mismatch
Single-strand break
Double-strand break
Interstrand crosslinks
Bulky adducts/Intrastrand crosslinks
Spontaneous alterations that require DNA repair
Oxidation
Hydrolysis
Methylation
Release of adenine or guanine bases
Depurination
Removal of amine group (NH2)
ex. C to U
Deamination
DNA repair mechanism for single-strand break/single base damage
Base-excision repair (BER)
DNA repair mechanism for Bulky lesions/Crosslinks
Nucleotide excision repair (NER)
DNA repair mechanism for base mismatch
Mismatch mediated repair (MMR)
DNA repair mechanism for double-strand break
Homologous Recombination (HR)
Non-homologous End-joining (NHEJ)
repairs damage to a single base caused by oxidation, alkylation, hydrolysis, or deamination.
Base excision repair (BER)
In BER, the damaged base is removed by a ________.
DNA glycosylase
In BER, the “missing tooth” is then recognized by an enzyme called ______, which cuts the phosphodiester bond. The missing part is then resynthesized by a DNA polymerase, and a DNA ligase performs the final nick-sealing step.
AP endonuclease
– recognizes bulky, cross-linking, helix-distorting lesions such as pyrimidine dimers and 6,4 photoproducts
Nucleotide excision repair (NER)
– corrects errors of DNA replication and recombination that result in mispaired (but undamaged) nucleotides.
Mismatch repair (MMR)
- is an autosomal recessive genetic disorder of DNA repair in which the ability to repair damage caused by ultraviolet (UV) light is deficient.
- Skin cancer.
- Due to failure in nucleotide-excision repair mechanism
Xeroderma pigmentosum
3 steps in Mismatch Repair (MMR)
- Recognition (mismatches)
- Degradation of error (exonucleases)
- Synthesis (correct sequence)
- Inherited disorder that increases the risk of developing colorectal cancer, endometrial cancer, ovarian cancer, etc.
- Due to mutations in MMR genes
Lynch syndrome/Hereditary non-polyposis colorectal cancer
– repairs oxidative damage, abasic sites, or errors of DNA topoisomerase
Single-strand break repair (SSBR)
- Precise repair pathway that requires a matching DNA sequence as a template
- It primarily uses the sister chromatid, a copy of the damaged DNA, for repair
- Active during S, G2 and M phase
Homologous recombination
- Simple and widely used mechanism
- Directly seals the broken ends of DNA without the need for a homologous DNA template
Non-Homologous End-Joining (NHEJ)
Significance of DNA repair
- Preservation of genetic information
- Prevention of diseases
- Protect against aging
- Response to environmental stress
- Contribution to the immune system (antibody diversity)
- Evolution and adaptation (allows for genetic variation)
