Replication_01

The S-phase of interphase

Region of the chromosome where replication initiates

One, their entire chromosome is replicated as a single replicon, starting at the oriC

• 2000 bp/min
• (much slower than the 50,000 bp/min with the bacterial replication fork)

• >6 hrs
• This suggests <15% of the replicons are active at a time

labeling newly synthesized DNA with BrdU, that allows them to be detected with a fluorescent labeled anti-BrdU antibodies

• 1. DNA replication is semiconservative
• 2. DNA replication is bidirectional
• 3. DNA replication is semidiscontinuous
• 4. DNA replication requires priming

helps to inactivate the gene because you are occupying more of the domain. A single cut has high fidelity liklihood for rejoining

"To date, this catalytically dead Cas9 (dCas9) has been employed outside the gene editing context, for example, for genome visualization, transcriptional regulation, epigenetic manipulation and investigation of chromatin composition" NIH

• destroying the function through degrading
• virus resistance in plants (many are RNA viruses)
• targeted RNA editing

The large size of Cas12 might disrupt the natural way that the protein its bound to operates

1 per fork, and one fork per bubble

GINS, CDC45, MCMs proteins at origin of replication

DNA ligase I

topoisomerase

TTAGGG

• semiconservative
• bidirectional
• semidiscontinuous
• requires priming

• Gyuarists and Wake (1973)
• Grew bacteria for several generations with 3H-Thymidine, which lightly radioactively labels DNA
• Higher does of 3H-thymidine added, which gives a dense radiographic label at the replication fork
• Radiography shows bidirectional replicaiton

Bidirectional

except for the colE1 plasmid of E.coli

• 5' to 3' writes
• NEVER in 3' to 5'

• Okazaki (1968) with pulse chase experiment.
• The fragments were fused to preformed longer DNA pieces with T4 DNA ligase

Lagging strand

• 3'-OH group
• Can be supplied by:

a preceding DNA fragment (as in nick translation or in rolling circle replication)

an RNA primer (since RNA polymerases can make RNA from scratch)

a DNA-binding protein covalently linked to a nucleotide (as in replication of some phages)

a preceding DNA fragment (as in nick translation or in rolling circle replication)

an RNA primer (since RNA polymerases can make RNA from scratch)

a DNA-binding protein covalently linked to a nucleotide (as in replication of some phages)

DNA replication begins with duplex melting and the creation of a “bubble” – a small region where parental strands have separated

Duplex melting relies on transcription and/or on plasmid-encoded trans-acting proteins (Reps) and primers for DNA synthesis are generated through processing (cleavage) of a transcript and/or by host- or plasmid-encoded primases

• As the bubble expands, replicating DNA begins to take on the q shape
• 

One strand of a dsDNA is nicked (or a primer is synthesized on ssDNA) and the 3’-end is extended

Intact DNA strand serves as a template

• The 5’-end is displaced
• 

• 5' -> 3' exonuclease
• needed for RNA primer remover for connecting Okazaki fragments
• DNA repair
• 

manifest their defects only in some conditions but are phenotypically normal under permissive conditions

• DNA polymerase III holoenzyme
• High fidelity: only 1 wrong neuclotide per 10,000,000 added
• proofreading tool
• Very fast: 2000 nucleotides/s

The holoenzyme is highly processive: > 500,000 dNTPs are added before the enzyme dissociates (compared to 20-50 dNTPs for pol I)

Replication of E. coli chromosome starts at oriC

oriC contains a number of functionally important DNA sequence motifs recognized by regulatory proteins (e.g., DnaA, Fis, IHF, and SeqA)

• DnaA protein is considered to be the initiator of chromosomal replication: multiple DnaA molecules assemble at oriC in a highly regulated manner
• 

characterized by localized DNA strand separation in the duplex unwinding element (DUE), an AT-rich region containing three 13-mer motifs (GATCTNTTNTTTT)


At initiation, when DnaA-ATP levels build up, it starts to bind to moderate-affinity DnaA boxes R5M and R3 and to two related low-affinity DNA motifs, named τ-sites (x2) and I-sites (x3)

Repressor Fis is displaced, and a histone-related activator protein IHF binds between R1 and τ-2 and bends DNA.

now the PRC forms

DnaA recruites DnaB-DnaC complexes, one for each strand of DNA

DnaB is the replicative helicase, a hexameric enzyme that unwinds the DNA: it migrates along the DNA in the direction of the replication fork movement and expands the region of single-stranded DNA

DnaC is the helicase loader that associates with DnaB in 1:1 ratio, brings DnaB to the DNA, and then dissociates

•     in an ATP-dependent manner
• 

both

Knockouts!

• Yeast-one hybrid
• (You cant start with a technique that requires prior knowledge of a protein)

protein that activates the initiation of DNA replication in procaryotes/bacteria

• hemimethylated due to semiconservative replication
• Dam (DNA-adenine methyltransferase) methalyates adenine base in the GATC sequence
• the oriC contains 11 copies of the palindromic sequence GATC where Dam will target
• this is a recruitment signal for SeqA

• protein that preferentialy binds to the hemimethylated GATC in oriC
• inhibits formation of new initiation complexes

Chromosomal replication must be highly regulated to ensure that the number of chromosomes in the cell remains constant

In E. coli, several mechanisms are in place to warrant that after a single round of chromosomal replication DNA does not replicate again

E. coli DNA is usually fully methylated, but right after replication (before the methylation marks are laid on the new strand) the DNA is hemimethylated

Hemimethylated DNA is only poorly recognized by DnaA (and DnaA is absolutely required for the initiation of replication and can bind with high affinity only to fully methylated oriC)

• The hemimethylated DNA  does not have affinity for DnaA
• SeqA binds to the hemimethylated sites to prevent transcription
• DnaA is bound to ADP after replication and needs to be converted back to ATP-bound DnaA

• Poor binding of the replication initiator protein DnaA to the hemimethylated OriC
• Strong binding of the replication repressor protein SeqA to the hemimethylated OriC
• Transcriptional repression of the DnaA gene due to the binding of SeqA to its promoter
• Transcriptional repression of the DnaA gene due to the binding of DnaA itself to its promoter
• Predominance of the inactive DnaA-ADP protein (as opposed to active DnaA-ADP) in the cell
• Doubling in the number of high-affinity DnaA boxes in the duplicated genome that sequester DnaA

• 
• Helicase (DnaB): hexameric protein that melts DNA at the replication fork using ATP hydrolysis for energy (1 ATP molecule for each base pair separated!)

Primase (DnaG): a monomeric protein that synthesizes RNA primers

• Single stranded DNA binding protein (SSB)-stabelized ssDNA

• 
• DNA Pol III holoenzyme: a mutisubunit protein that synthesizes DNA leading strand and Okazaki fragments on the lagging strand and consists of:

Pol III Core (aeq) x2 that makes and proofreads DNA: a is the polymerase, e is a proofreading exonuclease, and q stabilizes e

γ complex that loads β clamp onto the dsDNA

τ protein dimer that brings together two Pol III cores

Sliding clamp β, a ring-shaped homodimer of beta subunits that encircles dsDNA and “ties” Pol to the DNA

(a) The lagging strand template forms a loop through the replisome, and a new primer [2] has been made by the primase. A previously made Okazaki fragment [1] and the leading strand and its template are shown on the model

(b) The loop made by the lagging strand template grows by feeding through the replisome. The motion of the lower strand of the loop and dsDNA unwinding make room for the elongating Okazaki fragment [2]

(c) Elongating Okazaki fragment [2] reaches the primer [1] of the previous Okazaki fragment [1]

(d) Replisome releases the lagging strand loop, thus allowing primase to make a new primer [3]

In eukaryotes, DNA replication is regulated in the context of the cell cycle

Progression through the cell cycle is monitored at cell cycle checkpoints

• Decision whether a cell is ready to enter the S phase and replicate its DNA is made at the G1/S checkpoint (also known as the restriction checkpoint)
• At G1/S checkpoint, cyclin proteins trigger the process of DNA replication initiation by activating cyclin-dependent kinases (Cdk
• 

• G1/S checkpoint
• cyclin-dependent kinases are activated

• Origin Recognition Complex iis formed of six proteins
• bound to the ARS through cell cycle, but doesn't begin until lisencing factors are recruited to the origins

• protein-protein interaction
• phosphorylation
• ubiquitination
• exportation

travels along single strand of DNA and makes the replication bubble larger