Exam 4 DNA metabolism Flashcards
What is DNA metabolism?
Set of processes that achieve these tasks
- New DNA copy synthesized before each cell division
- Checking for errors that arise during/after DNA synthesis and repairing
- Segments of DNA rearranged within a chromosome or between two DNA molecules (recombination) which gives offspring a novel DNA
What are the 3 fundamental rules of replication?
- Replication is semiconservative (daughter strand made from parent strand, new DNA has parent and daughter strand)
- Begins at an origin and proceeds bidirectionally
- Synthesis of new DNA occurs in 5’ to 3’ direction and is semi-discontinuous
Meselson-Stahl experiment
- Proved Watson& Crick’s hypothesis of Semiconservative replication: new DNA has old (parent) strand and new (daughter) strand
- nitrogen used for synthesis of new dsDNA is equally divided between the two daughter genomes
Once replication begins, does it proceed in same or opposite directions?
- It is bidirectional (Has 2 replication forks)
- Both strands are replication simultaneously
- circular DNAs have extra loop
Can replication begin anywhere or does it begin in the same location?
Loops always initate/begin at same unique origin
- Inman’s experiment: denatured DNA at AT-rich regions -> “bubbles” that showed it starts at unique origin
What direction does synthesis proceed in?
- 5’ to 3’: always by addition of new nucleotides to the 3’ end (3’-OH)
- Leading strand: made continuously as replication fork moves
- Lagging strand: made discontinuously in short pieces (Okazaki) that’s later joined together
What are enzymes that degrade and create DNA segments?
- Nucleases: degrade nucleic acids (DNases: DNA, RNases: RNA)
- Exonucleases (from outside): cleave bonds that remove nucleotides from ends of DNA
- Endonucleases: cleaves within a DNA sequence
- Polymerases: build DNA strands (at least 5 DNA polymerases in E.Coli)
DNA elongation
- Template: parent strand
- Substrate: nucleoside triphosphates
- nucleophilic OH group at 3’ end of growing chain attacks a-phosphate of incoming trinucleotide (3’OH required and made more powerful by nearby Mg2+ ions)
- Pyrophasohate: good leaving group
DNA polymerase
- Needs a primer: shorter strand complementary to temple (contains 3’-OH to begin first reaction and made of RNA)
- Enzyme has pocket with 2 regions: insertion site (where incoming nucleotide binds), postinsertion site (newly made base pair resides when polymerase moves forward)
What is Processivity?
of nucleotides that DNA polymerase can add before dissociation (DNA poly. can add nucleotides or dissociate)
- Each polymerase has own processivity and polymerization rate
Geometry of Base pairing accounts for high Fidelity in E.Coli
DNA polymerase active site excludes base pairs with incorrect geometry but sometimes it’s still inserted - repair mechanisms fix this
What repair mechanisms fix errors during synthesis?
Errors during synthesis are corrected by 3’ -> 5’ exonuclease activity
- it proofread synthesis for mismatched base pair
- translocation of enzyme to next location is inhibited until the incorrect nucleotide is removed
5 DNA polymerases in E.Coli
- DNA Poly I: function is clean up (not ideal for replication bc slow replication fork and ow processivity (falls easily))
- DNA Poly III: replication polymerase
- DNA POly II, IV, V: DNA repair
DNA Polymerase I also has 5’ -> 3’ Exonuclease Activity (in addition to 3’ -> 5’ Exonuclease Activity)
- Moves ahead of enzyme and hydrolyzes things in its path
- Does nick translation (strand break moves along with enzyme)
(these happen in the Klenow fragment: domain that can be separated by Klenow fragment)
DNA Polymerase III
Replication Polymerase with 10 types of subunits (core domains linked by clamp-loader complex)
- Core domains interact with dimer of b-subunits that increase the processivity of the complex (by forming a sliding clamp that prevents dissocation)
What is required for E.Coli DNA Replication?
Replisome: made of over 20 enzymes and proteins
- Helicases (use ATP to unwind DNA), Topoisomerases (relieve stress caused by unwinding), DNA-binding proteins (stabilize separated strands), Primases (make RNA primers), DNA ligases (seal nicks between nucleotides on same strand)
Initiation of replication in E.Coli
- Beings at oriC site, contains highly conserved sequence elements made of 5 repeats of 9b-p sequence (R sites) that form binding site for DnaA (initiator protein)
- ## DNA unwinding element (DUE) A=T rich region
Regulation of Replication Initiation via Methylation
After replication: oriC methylated by Dam methylase (methylates N6 of A in GATC sequences)
- hemimethylated oriC seq. interact with plasma membrane using protein, SeqA
- SeqA dissociates and oriC sequences are released
- Dam fully methylates DNA allowing new DnaA to bind
How does elongation of the leading strand happen?
DnaG primase makes RNA primer, DnaG primase interacts with DnaB helicase but moves in the opposite direction to helicase, Dna Pol III adds nucleotides to the 3’ end of the strand (Pol III is linked to DnaB which is on the opposite DNA strand)
