Chapter 11 DNA Replication

DNA Polymerase

-uses nucleoside triphosphates and uses its catalytic activity to form a phosphodiester bond between them and the PRIMER to synthesize a new DNA strand -Must add phosphodiester bond between the 3' OH group of existing deoxynucleotide and 5' innermost phosphate group of new deoxynucleotide triphosphate, releasing the last two phosphate groups as pyrophosphate; therefore new strand grows in 5' to 3' direction using the template strand in the 3 to 5 direction synthesis is bidirectional

E. coli High fidelity reasons

1. Instability of mismatched pairs-bp most stable AT/GC (1/1000 error) 2. Configuration of the DNA polymerase active site -mismatches aren't energetically favorable, as a function of enzyme (1/1million error) 3. Proofreading exonuclease function of DNA polymerase (for both I and III)-repair is possible for mismatched (3' to 5' exonuclease)

Initiation of Replication in Euk.

ARs sequences, ORC and MCM helicase form preRC to complete DNA replication licensing which occurs only in G1 phase of cell cycle. Additional 22+ proteins are added in S phase that allow DNA synthesis to startand MCM helicase complex moves away from origin opening forms-replication can no longer re-initiate until next G1

Meselson-Stahl Experiment

Aimed to discover which model-conservative, semi-conservative or dispersive-was correct for DNA replication. A critical aspect of the experimental protocol was the use of isotopes to distinguish between the newly made strands versus the original strands. Due to density differences, the newly made strands were light, as opposed to the original parent strands that were heavy. Essentially they grew E. Coli in N isotope (heavy) for many generations and then stopped one day. The first generation would should different amounts of heavy or light depending on the mechanism. Results: After one round of replication in N14, dsDNA is all intermediate density After two rounds of replicaiton in N14, ½ of dsDNA is light and ½ is intermediate

oriC

aka origin of replication DNA sequence require for initiation in E. Coli (BACTERIA) DNA sequences in oriC include: AT-rich region DnaA boxes GATC methylation sites

E. Coli Regulation of DNA Replication

Bacterial cells regulate the DNA replication process by controlling the initiation of replication at oriC Regulation of initiation uses at least two mechanisms: regulation of DnaA functional concentration methylation of GATC sequences at oriC controls binding of DnaA protein

DNA ligase in E. coli

DNA ligase connects the Okazaki fragments by sealing the nick between fragments. This completes the synthesis of the lagging strand. , requires NAD+ in e. coli

Bacterial DNA Polymerases

DNA pol I and DNA pol III involved in DNA replication DNA pol-II, pol-IV, and pol-V are important in DNA repair

DNA polymerase I in E. Coli

DNA pol I will remove the RNA primers (5' to 3' exonuclease) and replace them with DNA (5' to 3' polymerase)

DNA Polymerase I and III in E. Coli

DNA polymerase (both pol III and pol I) can remove mismatched bases DNA polymerases can identify a mismatched nucleotide very near the 3' end and remove it from the daughter strand The enzyme uses its 3' to 5' exonuclease activity to remove the incorrect nucleotide It then resumes DNA synthesis in the 5' to 3' direction

pre-replciation complex (preRC) in euk

DNA replication begins with the assembly of the pre-replication complex (preRC), which includes: the origin recognition complex (ORC) binding to origins, six-subunits act as initiator of origin binding and then MCM helicase complex, which requires Cdt1 and Cdc6 (in G1 phase) completing DNA replication licensing

Licensing Functions

For "licensing", a complex of proteins binds to the origins of replication, including ORC, Cdt1, Cdc6 and MCM helicase. This process can only occur in the G1 phase of the cell cycle, prior to S phase (DNA Synthesis). As soon as DNA replication initiates at the origin in S phase, the "licensing" complex is changed/destroyed (the MCM helicase moves away; function of Cdt1 and Cdc6 is destroyed) so that it can no longer initiate replication. The "licensing" complex cannot reform until the next G1 phase, after mitosis is complete.

Bacterial DNA Replication

General Features Include: DNA synthesis begins at a single site termed the origin of replication Synthesis of DNA proceeds bidirectionally around the bacterial chromosome The replication forks eventually meet at the opposite side of the bacterial chromosome at termination sites Involves the following proteins: dnaA, dnaC, dnaB/DNA helicase, topoisomerase II/DNA gyrase, single-stranded binding proteins, primase, DNA pol III, DNA pol I, DNA ligase, Tus

dnaA proteins

In E. Coli, DNA replication begins by the binding of dnaA proteins to 5 sequences within the origin known as dnaA box sequences (HU an IHF also bind). This binding stimulates the cooperative binding of an additional 20 to 40 DnaA proteins to form a large complex The binding of dnaA causes the A/T rich region to denature into separate single-stranded regions. The dnaA proteins, with the help of the dnaC protein, then recruit DNA helicase proteins (dnaB protein) to oriC.

dnaC proteins

In E. Coli, The dnaA proteins, with the help of the dnaC protein, then recruit DNA helicase proteins (dnaB protein) to oriC.

Primosome

In E. Coli, a complex at the replication fork involving DNA helicase, primase, and several accessory proteins; this complex is known as a primosome.

DNA gyrase/topoisomerase II

In E. Coli, both DNA gyrase and topoisomerase II are the same thing. binds to the double-stranded DNA ahead of the replication fork to relax the positive supercoils that result from opening (denaturing) the double helix from DNA helicases/dnaB

Tus Protein

In E. Coli, termination utilization substance is a protein that binds to T1 and T2 to stop forks. Only one of the forks is stopped, and the other eventually meets. T1 stops counterclockwise T2 stocks clockwise

DNA helicases/dnaB

In E. Coli, the dnaA proteins, with the help of the dnaC protein, then recruit DNA helicase proteins (dnaB protein) to AT-rich bubble in the oriC. DNA helicases promote strand separation within the oriC region and beyond. To do so, DNA helicases bind to single-stranded DNA and travel along the DNA to keep the replication fork moving in the 5' to 3' direction, disrupting hydrogen bonds along the way and creating two replication forks.

Replisome

In E. Coli, two DNA polymerases (i.e., dimeric DNA polymerase) are associated with the primosome so that DNA helicase, primase, and the DNA polymerases move as a single unit along the replication fork. The DNA lagging strand must loop with respect to the DNA polymerase synthesizing it, allowing it to synthesize in 5' to 3' direction and move toward the replication fork at the same time.

Catenanes

In E. coli, DNA replication results in two intertwined DNA strands, termed catenanes. These are separated by topoisomerases.

Flap endonuclease

In eukaryotes RNA primers are removed by a separate protein called Flap endonuclease. On the lagging strand, the delta DNA polymerase elongates a Okazaki fragment on the 3' end past the RNA primer of the adjacent Okazaki fragments, which causes a short flap to form. The Flap endonuclease removes the RNA primer and the short flap (cannot remove long flaps). If a flap is too long, Dna 2 nuclease/helicase shortens the flap enough for Flap endonuclease to remove it. DNA ligase seals the DNA fragments together.

DNA polymerase III in E. Coli

In leading strand: DNA pol III attaches nucleotides toward the opening of the replication fork In lagging strand: DNA pol III uses the RNA primers to synthesize small DNA fragments (1000 to 2000 nucleotides each) -- Okazaki fragments DNA pol-III synthesizes both the leading and lagging strands, synthesizing only in the 5' to 3' direction During replication, it is a processive enzyme, which means that it glides along the template strand as it synthesizes a new daughter strand and does not fall off.

MCM helicase

In the G1 phase of the cell cycle, prior to S phase and the initiation of DNA replication, ORC and then MCM bind to origins in a process called DNA replication licensing (see PowerPoint). This binding of MCM is linked to the cell cycle, since it requires specific cell cycle proteins, Cdt1 and Cdc6, which are present in G1 phase and then degraded before S phase. Thus, only origins with MCM bound in G1 phase will initiate DNA replication during S phase, and so origins can only initiate DNA replication once during a cell cycle.

ARS sequences

In yeast, ARS (autonomously replicating sequence) elements are thought to play the analogous role that oriC plays in E. coli. ARS consists of about 50 bp containing high A-T bases and ARS consensus sequences and additional elements that enhance origin function. Many are located throughout the DNA so there are many origins of replication

Leading Strand

One RNA primer is made at the origin by DNA primase DNA pol III (E. coli enzyme) attaches nucleotides in a 5' to 3' direction (nucleotides added to free 3' hydroxyl group) continuously toward the opening of the replication fork

Leading Strand DNA Replication Synthesis in E. Coli

One RNA primer is made at the origin by DNA primase DNA pol III attaches nucleotides toward the opening of the replication fork helicase continues to unwind DNA of double helix DNA at fork topoisomerase II makes negative supercoils to relieve positive supercoils

dnaA Regulation of DNA Replication

One way to prevent premature re-initiation is the decrease in the relative concentration of active dnaA protein to DnaA box sequences in oriC, immediately following initiation ATP-bound dnaA protein binds cooperatively to all dnaA boxes for initiation. dna A protein is ADP bound after initiation therefore the relative concentration of dna A protein to dnaA boxes is low after initiation. The complex forms less often because dnaA is less functional.

Lagging Strand

Synthesis is also in the 5' to 3' direction actual synthesis goes away from the replication fork Many RNA primers are sequentially synthesized at the fork by DNA primase DNA pol III (E. coli enzyme) uses the RNA primers to synthesize small DNA fragments (1000 to 2000 nucleotides each) These are termed Okazaki fragments after their discoverers DNA pol I (E. coli enzyme) will remove the RNA primers (5' to 3' exonuclease) and replace them with DNA (5' to 3' polymerase) DNA ligase catalyzes a phosphodiester bond between the phosphates of adjacent nucleotides to seal the remaining nick in the sugar-phosphate backbone

Telomere Replication Cycle

Telomerase adds DNA sequence repeats at the 5' end of the new strand allowing for a primer to be placed (by Alpha polymerase) and that section replaced. 1) Binding-telomerase binds by complimentary bind of its RNA sequence to the 3' overhang of of the lagging template strand. 2) Polymerization--telomerase adds 6 nucleotides to the 3' end of the template strand 3) Translocation-telomerase moves to the right and adds another repeat if necessary 4) Priming-Complementary strand is made by primase 5) Polymeraization-DNA polymerase can now use the primer strand to add DNA sequences to the 3' end of the primer at the 5' end of the new strand. 6) Ligation-ligase binds the two strands together.

Telomerase

Telomerase contains protein and RNA: The RNA is complementary to the DNA sequence found in the telomeric repeat-this allows the telomerase to bind to the 3' overhang RNA acts as a template for telomerase to synthesize extension to 3' overhang synthesis of DNA using RNA template done by reverse transcriptase

Isolation of Mutants

The isolation of dna mutants was important in several ways The mutants identified genes that were required in vivo for replication It allowed for the identification and biochemical analysis of the proteins that were defective in the mutants 3. It allowed for the mapping of these mutations/genes along the E. coli chromosome 4. It provided an important starting point for the subsequent cloning and sequencing of these genes

Telomere

The telomere is a complex of telomeric sequences within the DNA and proteins that are bound to these sequences. Telomeric sequences consist of a moderately repetitive tandem array and a 3' "overhang" region that is 12 to 16 nucleotides in length (several guanines and thymines.

Termination of Replication in E. Coli

a pair of termination sequences called ter sequences T1 stops counterclockwise forks, T2 stops clockwise The protein tus (termination utilization substance) binds to T1 and T2 to stop forks DNA replication ends when oppositely advancing forks meet (usually at T1 or T2) DNA ligase covalently links DNA strands DNA replication often results in two intertwined molecules Intertwined circular molecules are termed catenanes These are separated by the action of topoisomerases

Lagging Strand DNA replication Synthesis in E. Coli

actual synthesis goes away from the replication fork Many RNA primers are sequentially synthesized at the fork by DNA primase DNA pol III uses the RNA primers to synthesize small DNA fragments (1000 to 2000 nucleotides each) -- Okazaki fragments DNA pol I will remove the RNA primers (5' to 3' exonuclease) and replace them with DNA (5' to 3' polymerase) DNA ligase catalyzes a phosphodiester bond between the phosphates of adjacent nucleotides to seal the nick in the backbone helicase continues to unwind DNA of double helix DNA at fork topoisomerase II makes negative supercoils to relieve positive supercoils

Single Stranded Binding proteins

aka SSB proteins in E. Coli bind to both of the single strands of parental DNA and prevent them from reforming a double helix. After this the DNA is ready to be replicated

Telomere replication

because polymerase need a primer to begin synthesis and add nucleotides to the 3' end, at the 5' end of a new strand (3' end of template) a primer cannot be replaced because there is no 3' OH group to prime the synthesis of that section. Chromosomes would continue getting shorter. This is solved using telomerase which adds DNA sequence repeats at the 5' end of the new strand allowing for a primer to be placed (by Alpha polymerase) and that section replaced.

DNA Primase

in E. Coli, DNA polymerase is not able to initiate synthesis de novo; it must add to a pre-existing nucleic acid "primer". In replication, the initial primer is a short RNA molecule that is synthesized by DNA primase. Leading strand contains only one RNA primer in the oriC region, while the lagging strand contains multiple RNA primers that are eventually removed.

Dam methylase Regulation of DNA replication

involves the GATC sites within oriC. Following DNA replication, the newly made strands are hemimethylated. Dam methylase enzyme (DNA adenine methyltransferase) recognizes the 5'GATC3' sequence and adds a methyl group onto adenine many minutes after DNA replication The hemimethylated DNA is NOT ABLE to initiate DNA replication until after it has become fully methylated which may take several minutes. This is because dnaA protein cannot access dnaA boxes efficiently

DNA replication initiation proteins/sequences in E. Coli

oriC and AT Rich regions dna A dna C dna B/DNA helicase topoisomerase SSB

Eukaryotic DNA Polymerases

α, ε, δ are DNA replicating polymerases, while γ functions in mitochondria to replicate mitochondrial DNA. α, initiates DNA replication-because it has a primase as one of its subunits-on both leading and lagging strand. About 10 RNA then 20-30 DNA ε, synthesizes the leading strand during S phase, after switching in for alpha δ, synthesizes the lagging strand during S phase, after switching in for alpha

e. coli DNA polymerase III subunits

α, ε, θ, β, γ α is the DNA catalytic subunit β clamp protein (Table 11.2), which encircles the DNA. The clamp is put on the DNA by the clamp-loader protein complex. With the B subunit the DNA polymerase II can synthesize 750 n/s, without only 20 n/s. γ subunit is the clamp-loader protein, needed for beta-clamp to initially clamp onto DNA