Unit 9.1 Flashcards
Fill in the blank: DNA is the only molecule that_______ b/c it is the database for information
Gets repaired
What are the 3 R’s of DNA metabolism
- Replication
- Repair
- Recombination
what is replication, repair and recombination
replication: the duplication of genetic information
repair: maintenance of information
recombination: cleaving and rejoining DNA for repair and shuffling information
What form of transmission is DNA passed to the next generation? Horizontal or vertical?
Vertical transmission- passed to the next generation
not horizontal transmission b/c that involves passing to another cell
What does DNA replication require
Fidelity
- faithful copying of each strand
- requires proofreading
What happens if defects in DNA replication are left unrepaired?
they lead to mutations and are passed on to the next generation.
describe DNA replication
- uses 1 strand as template
- Semi conservative mechanism
- DNA polymerases (actually carry out the synthesis) and others responsible for replication
- need at least 20 proteins to carry out replication
- bidirectional replication (2 replication forks, 1 origin)
Meselson-stahl experiment
- uses sedimentation equilibrium centrifugation
- semiconservative mechanism
describe replication forks
- where proteins assemble for replication
- assembly points (aka replisome)
- y shaped structures
what are the required steps for replication?
1st. melt/denature the double helix.
- this exposes the bases for selection
2nd. need primer
- a short piece of nucleic acid that hybridizes with the template
- provides the free -OH group on the 3’ end
3rd. add new nucleotides to the 3’ OH end
- hydrogen bonds between template and new nucleotide
- phosphodiester bonds to create a new backbone in the newly synthesized strand
4th. release torsional stress
- topoisomerases
Describe DNA polymerases
- catalyze the addition of complementary nucleotide to the 3’ end of the DNA strand
- requires: DNA template, Primer (DNA or RNA), and deoxynucleotides (can be dNTP)
- need Mg2+ b/c to orient the molecules the proper distance for binding
- synthesis is 5’ to 3’
- template is read 3’ to 5’
What happens during the DNA polymerase rxn
- Mg2+ is needed to position the nucleotide in the right orientation for binding
- ## pyrophosphatase cleaves the pyrophosphate
describe proofreading activity
- works backwards 3’ to 5’
- intrinisic 3’ to 5’ exonuclease activity (cuts at the end of the strand.
What determines fidelity?
- proofreading activity (exonuclease, endonuclease)
- polymerase activity ( template-directed enzymes, priming produces more accuracy which is why DNA polymerase is more accurate than RNA polymerase)
What is processive in terms of polymerases?
polymerases synthesize several bonds at the same time before releasing the substrate (strand)
What triggers the proofreading function?
- incorrect base does not fit properly in active site stalling the polymerase and inducing a conformational change
What happens if the polymerase proofreading activity is not triggered?
- replication mismatch repair can switch out the base
Who has more DNA polymerases prokaryotes or eukaryotes?
- Eukaryotes
describe the DNA polymerases in prokaryotes
DNA Polymerase I - primer removal, fills gaps in lagging strand
DNA Polymerase II- DNA repair
DNA Polymerase III- major replicative polymerase
describe the DNA polymerases in eukaryotes
- Polymerase for initiating replication (contains primase)
- Polymerase for DNA repair
- Polymerase for major replicative polymerase (lagging strand)
- Polymerase for major replicative polymerase (leading strand)
Describe helicase
- catalyzes double strand separation of partially melted dsDNA
- helicase requires ATP hydrolysis for conformational changes to bind to single strand
- Binds to ssDNA. initial denaturation is done by primosome
- moves along and separates the double helix
What do single-strand DNA binding proteins do?
- stabilize ssDNA by preventing secondary structure from forming
- protect ssDNA from degrading
- keep bases accessible for polymerases
- aka helix destabilizing proteins
describe primase
responsible for primer synthesis
- it is RNA polymerase in prokaryotes
- it is RNA-DNA primer in eukaryotes (Primase- Pola complex)
it interacts with helicase
lagging strand requires multiple priming
-primers are replaced by DNA. RNAse removes it (RNA degrading enzyme)
describe DNA ligase
- seals ssDNA breaks in backbone during replication and repair
- forms phosphodiester bond between 3’ OH and 5’ Phosphate end
- used in dsDNA
- requires ATP
describe the lagging strand synthesis process
- new RNA primer synthesis by DNA primase
- DNA polymerase adds to new RNA primer to start a new Okazaki fragment
- DNA polymerase finishes DNA fragment
- old RNA primer is erased and replaced by DNA
- sealing by DNA ligase joins new Okazaki fragment to the growing chain
describe polymerase sliding clamp
- clamps the DNA polymerase to keep it from sliding off the template
- enhances polymerase processivity by preventing dissociation of polymerase
is initially loaded to the template by a clamp loader enzyme
describe the sliding clamp loader
- displaces primase after primer synthesis
- key organizer of replisome
- ATP binding to clamp loader opens sliding clamp
- open clamp moves on to DNA
- ATP hydrolysis locks sliding clamp around DNA and releases clamp loader
- DNA polymerase binds to sliding clamp
describe E.coli DNA polymerase holoenzyme
- it’s 2 DNA Pol core domains linked through central clamp loading complex
- interacts with helicase through subunits
describe initiation of replication
- occurs at special DNA sequences (origins)
- is a single site in prokaryotic chromosomes
- is multiple sites in eukaryotic chromosomes
- act as assembly sites for initiator proteins that initiate synthesis
- tightly regulated. the only regulated site in DNA synthesis
- where you would act if you wanted to stop replication
- region is AT rich because melting is easier since there are only two H bonds
What is the primosome in eukaryotes?
Helicase and primase complex
