DNA Replication
What is the function of the sliding clamp in DNA replication?
Holds DNA polymerase in place during strand extension.
Lagging Strand (discontinuous strand) (definition)
In DNA replication, the new strand of DNA that is synthesized discontinuously (as a series of short pieces that are later joined) in a direction moving away from the replication fork. Also called discontinuous strand.
Leading Strand (Continuous Strand) (definition)
In DNA replication, the new strand of DNA that is synthesized in one continuous piece in a direction that follows the replication fork. Also called continuous strand.
What protein recognizes and binds to the origin of replication sequences?
Initiator Proteins.
Replication Fork (definition)
The Y-shaped site at which a double-stranded molecule of DNA is separated into two single strands for replication.
Origin of Replication (definition)
The site on a chromosome at which DNA replication begins.
In which direction is the lagging strand synthesized?
Away from the replication fork.
DNA Replication: Synthesis of the Lagging Strand
*1. A Primer is added:* Primase synthesizes an RNA primer. *2. Okazaki Fragments are synthesized:* DNA polymerase III works in 5'—> 3' direction, synthesizing Okazaki fragment of the lagging strand. Every Okazaki fragment needs to be primed by Primase. *3. DNA Polymerase I replaces primers:* DNA polymerase I removes ribonucleotides of the primer, and replaces them with deoxyribonucleotides in the 5' —> 3' direction. *4. DNA Ligases closes the gaps in the backbone:* Once the RNA primer is removed and replaced by DNA, DNA Ligase catalyzes the formation of a phosphodiester bond between the adjacent fragments.
DNA Replication Steps: Synthesis of the Leading Strand
*1. DNA is opened, and unwound and primed. A replication bubble is formed at the origin of replication.* A. *DNA Helicase:* Once a specific set of proteins recognizes the origin of replication, DNA Helicase breaks the hydrogen bonds between the base pairs in that location and opens the double helix there. This reaction causes the two strands of DNA to separate at the replication fork. B. *Single-strand DNA-binding proteins (SSBPs):* SSBPs attach to the separated strands to prevent them from snapping back into a double helix. C. *Topoisomerase:* The unwinding of DNA strands at the replication fork creates twists farther down the helix. The twisting induced by helicase is relaxed by Topoisomerase. D. *Primase:* Primase is a type of RNA polymerase, it synthesizes the short stretch of RNA that acts as a primer for DNA polymerase. The primer provides DNA polymerase with a 3′ hydroxyl (-OH) group that can be linked to a deoxyribonucleotide to form a phosphodiester bond. *2. Synthesis of Leading Strand:* A. *DNA Polymerase III:* Once a primer has been laid down on a single-stranded template, DNA polymerase III begins adding deoxyribonucleotides in the 5′—> 3′ direction of the leading strand. It adds deoxyribonucleotides to the 3′ end of the old DNA strand. The enzyme moves into the replication fork, which is unwound ahead of it. B. *Sliding Clamp:* Part of DNA Polymerase III forms a ring—the Sliding Clamp—that surrounds the DNA, and another part grips the DNA strand. Deoxyribonucleotide addition is catalyzed at an active site in it's groove.
RNA Polymerase (definition, role in DNA Replication)
*Definition*: An enzyme that catalyzes the synthesis of RNA from ribonucleotides using a DNA template. *Role:* In DNA Replication, Primase, a type of RNA Polymerase, synthesizes the short stretch of RNA that acts as a primer for DNA polymerase.
Deoxyribonucleoside Triphosphate (dNTP) (definition, role, energy)
*Definition:* A monomer used by DNA polymerase to polymerize DNA. Consists of the sugar deoxyribose, a base (A, T, G, or C), and three phosphate groups. *Role:* DNA synthesis (in DNA Replication) involves a condensation reaction that forms a phosphodiester bond between the 3′ carbon on the end of a DNA strand and the 5′ carbon on an incoming deoxyribonucleoside triphosphate (dNTP) monomer. *Energy:* Polymerization reactions generally are endergonic, meaning they require an input of energy. The potential energy of the deoxyribonucleotide monomers is first raised by reactions that add two phosphate groups to form dNTPS.
Single-Strand DNA-Binding Proteins (SSBPs) (definition, role)
*Definition:* A protein that attaches to separated strands of DNA during replication, preventing them from re-forming a double helix. *Role:* Attach to the separated DNA strands during DNA Replication to prevent them from snapping back into a double helix.
Primer (definition, role)
*Definition:* A short, single-stranded RNA molecule that base-pairs with a DNA template strand and is used as a starting point for DNA synthesis by DNA polymerase. *Role:* The primer provides DNA polymerase with a 3′ hydroxyl (-OH) group that can be linked to a deoxyribonucleotide to form a phosphodiester bond. Primase (an RNA Polymerase) synthesizes the primer.
DNA Helicase (definition, role)
*Definition:* An enzyme that breaks hydrogen bonds between nucleotides of DNA, "unzipping" a double-stranded DNA molecule. *Role:* In eukaryotes, the proteins that initiate DNA replication are under tight control by cell-cycle regulatory proteins. Once a specific set of proteins recognizes the origin on a bacterial or a eukaryotic chromosome, the enzyme DNA helicase breaks the hydrogen bonds between the base pairs in that location and opens the double helix there. This reaction causes the two strands of DNA to separate at the replication fork.
DNA Ligase (definition, role)
*Definition:* An enzyme that joins pieces of DNA by catalyzing the formation of a phosphodiester bond between the pieces. *Role:* Due to the lagging strand having to be synthesized with Okazaki Fragments, after Polymerase I removed the primers, DNA Ligase catalyzes the formation of a phosphodiester bond between the adjacent fragments. It closes the gaps in the chain.
Topoisomerase (definition, role)
*Definition:* An enzyme that prevents the twisting of DNA ahead of the advancing replication fork by cutting the DNA, allowing it to unwind, and rejoining it. *Role:* During DNA Replication the unwinding of DNA strands by DNA Helicase at the replication fork creates twists farther down the helix.
DNA Polymerase (definition, role, restrictions, types)
*Definition:* Any enzyme that catalyzes synthesis of DNA from deoxyribonucleoside triphosphates (dNTPs). *Role:* It polymerizes deoxyribonucleotide monomers into DNA. It catalyzes DNA synthesis. *Restrictions:* It can only work in one direction. DNA polymerase (1) works only in the 5′ —> 3′ direction along a single-stranded template and (2) requires a 3′ end (non-template) to extend from (i.e. add deoxyribonucleotides). *Types:* - *DNA Polymerase III:* Extends the leading Strand during DNA Replication. Extends Okazaki Fragments in the lagging strand during DNA replication. - *DNA Polymerase I:* Removed the RNA primer and replaces it with DNA during lagging strand synthesis of DNA Replication.
Okazaki fragments (definition, role, structure)
*Definition:* Short segment of DNA produced during replication of the lagging-strand template. The Okazaki fragments are eventually linked together to produce the lagging strand in newly synthesized DNA. *Role:* The lagging strand is made in Okazaki Fragments because, as the fork moves forward, the DNA polymerase (which is moving away from the fork) must come off and reattach on the newly exposed DNA. *Structure:* Okazaki fragments in eukaryotes are shorter than in bacteria, and the way primers are removed is different, but the mechanism of synthesizing Okazaki fragments that are joined into an unbroken chain of DNA is the same.
Replisome (definition, structure)
*Definition:* The macromolecular machine that copies DNA; includes DNA polymerase, helicase, primase, and other enzymes. *Structure & Function:* - Most of the enzymes in DNA Replication in actuality are joined into the replisome, a large macromolecular machine. - In E. coli, the replisome contains two copies of DNA polymerase III that coordinately engage in DNA synthesis. The lagging strand loops around the complex, allowing the replisome to move as a single unit as it follows the replication fork. - Once the DNA polymerase on the lagging strand completes synthesis of an Okazaki fragment, it releases the DNA and reassembles on the most recently synthesized primer.
Nick (definition)
A nick is a break in newly synthesized DNA, where the phosphodiester bond between two adjective nucleotides is missing. Nicks arise in DNA replication after DNA Polymerase I removes the RNA primer and replaces it with DNA. Ligase creates the missing bond, sealing the nick.
Are initiator proteins expressed constitutively?
No, the cell doesn't need to replicate DNA all the time. It only replicates DNA when the cell divides.