Replication Flashcards
What are inferred types of models for DNA Replication?
Conservative: original model is conserved when synthesizing a completely new double stranded DNA
Semiconservative: each parental “old” strand serves as a template for synthesis of “new” daughter DNA strand
Dispersive: original molecule is replicated and each strand in the daughter molecule is a combination of synthesized and parent DNA
What is the Meselson and Stahl experiment?
Two groups of cells of E. coli grown, one with nitrogenous bases of N15 isotope and the other with N14 isotope. N15 E. coli is added to N14 medium. DNA is extracted and spun via density gradient centrifugation.
How does the Meselson and Stahl Experiment determine the correct model of DNA replication?
After the first replication, density gradient medium reveals a single band of N14/N15 (ruling out conservative), after second replication medium reveals two bands for N14 and N15 (ruling out dispersive)
What is the bidirectional progression of replication of E. coli?
- Replisome binds to DNA and initiates synthesis at replication forks
- Replication forks continue synthesis in opposite directions
- Replication forks hit terminus of replication and collide, releasing two chromosome copies
How is the replication of E. coli’s chromosome discovered?
E. coli was fed radioactive precursor (thymine) and was exposed to film to demonstrate oriC (initiation site) and ter (termination. site) as well as the replication bubble.
What is a consensus sequence
a common nucleotide sequence or amino acid sequence found in highly conserved regions of DNA or RNA or proteins
What are the regions on the E. coli oriC?
- AT rich regions
- Sites for DnaA binding
- Methylation sites
- Sites for FIS and IHF proteins
What are the steps of initiation of replication?
- dnaA binds to high affinity dna A binding sites, which is used as an anchor to cooperatively bind ATP-dnaA complexes to the low affinity AT-rich region
- Topological stress breaks H-bonds and unwinds AT-rich region with the helps of HU, which provides a higher affinity to ATP-dnaA molecules
- ssb proteins stabilize single stranded DNA which prevents helix formation
- 6 monomers of dnaC recruits 6 monomers of dnaB onto each single strand
- dnaB monomers form a hexameric ring around each strand and dnaC monomers dissociate
- After helixase moves outward a bit, primase is loaded behind each helicase
- Sliding clamp is loaded between helicase and DNA polymerase III, while ATP-dnaA hydrolyzes to ADP-dnaA which promotes dissociation
- Primase lays down RNA primers that has 3’ OH ends that will be recognized by DNA polymerase III for DNA synthesis
What is a kilobase pair and a megabase pair?
kbp: 1,000 base pairs
mbp: 1,000,000 base pairs
What are the levels involved in supercoiling?
lvl 1 - double helix
lvl 2 - supercoils within the double helix via DNA binding proteins
What is the genetic map of E. coli?
1%: tRNa, rRNA, ncRNA
88%: enzyme encoding genes that may or may not be clustered together in an operon
What are plasmids?
Typically circular non-essential genes in addition to the chromosomes in a bacteria/archaea’s genome that encodes beneficial genes to host cell.
What are some of the beneficial genes plasmids encode?
Virulence, antibiotic resistance, bacteriocins, nitrogen fixation, bioremediation
What are the features of plasmid R100
- tra region (conjugation functions)
- IS10 (transposable elements)
- resistance determinants (resistance against heavy metals, antibiotics)
What are the precursor molecules of nucleotides that will be used in DNA synthesis?
Deoxynucleotide 5’triphosphate (dNTP). Added to the next molecules 3’ OH, and two terminal phosphates are removed.
What is the direction of DNA synthesis?
Starts with first 5’ phosphate, ends with last 3’ sugar
What protein does gene gyrAB encode?
DNA gyrase: cleaves strands of DNA and reseals the ends to replace supercoils ahead of the replisome
What protein does gene dnaA encode?
Origin binding protein: binds to oriC to open double helix
What protein does dnaC encode?
Helicase loader: loads helicase at origin
What does protein dnaB encode?
Helicase: unwinds double helix at replication fork
What protein does gene ssb encode?
Single strand binding protein: prevents single strands from annealing
What protein does gene dnaG encode?
Primase: primes new strands of DNA
What protein does gene dnaN encode?
Sliding clamp: holds DNA Polymerase III onto DNA
What protein does holA-E encode?
Clamp loader: loads DNA Polymerase III onto sliding clamp
What protein does dnaX encode?
Tau subunit: holds two core enzymes for leading and lagging strand
What protein does dnaE encode?
Polymerase subunit: involved in strand elongation
What protein does dnaQ encode?
Proofreeding subunit
What protein does polA encode?
DNA polymerase I: excises RNA primer and fills in gaps pacman style
What protein does lig A encode?
DNA ligase: seals gaps in DNA
What protein does the gene tus encode?
Binds terminus and blocks progress of the replication fork
What protein does the gene parCE encode?
TopoisomeraseIV: unlinks interlocked circles
What does helicase do?
Hydrolyzes ATP to separate strands in advance of a replication fork, causing supercoiling, putting the molecule under torsion
What is the difference between negative and positive supercoiling?
Negative: DNA is twisted about axis in the left hand direction (counter clockwise)
Positive: DNA is twisted about axis in the right hand direction (clockwise)
What is the difference between the leading strand and lagging strand orientation and synthesis?
Leading: orientation of template strand is oriC (3’) to fork joint (5’) and leading strand synthesis proceeds discontinuously towards the fork joint
Lagging: orientation of template strand is fork joint (5’) to oriC (3’) and synthesis proceeds discontinuously towards the oriC
What are the steps of synthesis of Okazaki fragments?
- RNA primer is set by primase
- DNA is synthesized by DNA polymerase III
- Pol III approaches end of fragment and primase makes new primer and dissociates, while Primosome loads new beta clamp to the second fragment
- Pol III completes okazaki fragment, dissociates from old B-clamp, associates with new B-clamp, and begins synthesis of new fragment
- DNA polymerase I removes RNA primer and synthesizes DNA in its place.
- DNA ligase completes last phosphodiester bond via ATP hydrolysis
What are mechanisms used to reduce error rate?
- Incorporation of dNTP by DNA pol I and III to match base pairing accurately
- DNA polymerase I has 5’ to 3’ exonuclease activity to remove potentially mismatched primers
- Methyl directed mismatch repair
- 3’-5’ exonuclease activity of DNA pol I and III
How does DNA polymerase III and DNA polymerase I proofread?
DnaQ of Polymerase III and DNA polymerase I are able to sense distortions from mis-base pairing in the helix
How is chromosomal replication terminated?
- Replication forks meet at ter site
- Tus protein binds to ter and stops replication forks
- Topoisomerase IV decatenates interlocked chromosomes
What are the two ways chromosome replication is controlled?
- Dna-ATP is blocked/inactivated which prevents the unwinding of the helix
- Newly replicated hemimethylated GATC sequences in the in the oriC region are methylated by DNA adenine methylase
What are the mechanisms that control the activation of DnaA-ATP?
- Creating competition for oriC binding
- repression of dnaA expression
- reduction of DnaA-ATP levels
What does SeqA do?
SeqA protein blocks hemimethylated oriC sequences which preventing the re-firing of oriC by DnA-ATP after replication begins.
How is repression of dnaA expression controlled?
dnaA gene is inhibited by DnaA-ATP binding to DnaA boxes on the dnaA gene located in the promotor region.
How are DnaA-ATP levels reduced?
ATPase HdaA associates with DNA in the proximity of the replisome and promotes hydrolysis of DnaA-ATP to DnaA-ADP in a replication-dependent manner.
How long does it take for genome replication and cell division (generation) in low and rich nutrient conditions?
Low: replication (40mins), division (60 mins)
Rich: replication (20 mins), division (40 mins)
What happens to the chromosomes when an E. coli cells is replicating at its constant and maximal speed?
New replication rounds are iniitated before the previous round is cpmoleted, and newly synthesized DNA is methylated which prevents SeqA to bind and allows DnaA-ATP to initiate another replication round.
What is the MukBEF complex?
Protein complex binding to the nucleoid and localizes to distinct regions to assist tomopisomerase IV with the separation of the replicated chromosomes. Together they form the structural maintenance of chromosome (SMC) complex
How does the what proteins re involved in the divosome and how does it operate?
MinC, Min D: forms spiral structure on the inner surface of cytoplasmic membrane along cell axis, inhibits cell division
Min E: moves to each pole, pushing MinC and Min D aside
FtsZ: forms ring at lowest concentration of Min proteins via GTP hydrolysis
What does FtsZ attract?
ZipA: anchors Fts ring to cytoplasmic membrane for stability
FtsA: ATPase that recruits other FtsA, FtsI, and FtsK and proteins required for peptidoglycan synthesis. it also connects ring to cytoplasmic membrane
FtsK: translocates chromosome at division septum and regulate decatenation of chromosomes with topoisomerase IV
MukB: interacts with topoisomerase IV to remove catenanes before DNA is condensed
