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Origin of replication

Place where DNA replication begins (DNA unwinds from there).

Replication fork

For each origin of replication, there are two "replication forks" at which new DNA is formed.

DNA replication: Directionality

5' → 3'
(3' hydroxyl at the end of the growing chain is a nucleophile which attacks the phosphorous on the nucleotide to be added; pyrophosphate eliminated and phosphodiester bond formed.)

Leading strand

Formed continuously

Lagging strand

Formed discontinously (in 1000 to 2000 nucleotides, typically).

Okazaki fragments

Fragments that result from discontinuous DNA replication on lagging strand.

DNA ligase

Enzyme that links Okazaki fragments of lagging strand.

Pol I (E. coli)

Single polypeptide chain polymerase. Repairing and "patching" DNA. Removes RNA primers.

Pol II (E. coli)

Multi-peptide chain polymerase. Repair enzyme.

Pol III (E. coli)

Made of 10 subunits; core enzyme responsible for DNA polymerization. "Clamps" on parent DNA and slides along it during replication. Main polymerizing enzyme in prokaryotes.

Primer

Short strand of RNA that first must be added to DNA to be replicated; polymerases can only add DNA nucleotides if this primer is present. (both prok. and euk.)

3' → 5' exonuclease activity

Proofreading function. Done one nucleotide at at time. All three polymerases (I, II, III) can do this.

5' → 3' exonuclease activity

Repair function. Done several nucleotides at a time; also how RNA primers are removed. Only Pol I can do this.

Helicase

Enzyme that promotes unwinding of DNA for replication. (Both proks. and euks.)

Single-strand binding protein

Stabilizes single-stranded DNA during replication by binding tightly; protects from hydrolysis by nucleases in the cell.

Primase

Enzyme that adds RNA primer to DNA to be replicated.

Nick translation

Pol I uses its 5' → 3' exonuclease activity to remove RNA primers or DNA mistakes missed by proofreading, then fills in behind with its polymerase activity.

Mismatch repair

Enzymes recognize that two bases are incorrectly paired, and remove mismatch (occurs when damage escapes normal exonuclease activity of Pol I and Pol III). Polymerases then fill in gaps. Enzymes know which is the parent strand because prokaryotes methylate DNA at certain locations.

Base excision repair

DNA glycolase removes base damaged by oxidation or chemical modification; AP endonuclease removes sugar and phosphate. Excision exonuclease removes more bases. Pol I fills in gap; DNA ligases seals phosphodiester backbone.

Nucleotide-excision repair

Common for DNA damage done by UV or chemicals. DNA removed by ABC excinuclease; DNA Pol I and DNA ligase fill in gap.

Complications in eukaryotic DNA replication vs. prokaryotic (3)

1. Multiple origins of replication
2. Timing must be controlled to that of cell division
3. More proteins and enzymes involved

When does DNA replication take place in cell cycle?

S phase

Replicators

The origins of replication (multiple) in eukaryotes. Specific DNA sequences between genes.

Replicons

Zones where replication is occurring in eukaryotes (500 - 50,000 bp's).

What phase must cell reach to be competent to undergo DNA replication?

G₁

ORC

Origin recognition complex - multi-subunit protein that initiates replication. Bound to DNA thorugh cell cycle, but serves as attachment site for other proteins that control replication. (eukaryotes only)

RAP

Replication activator protein. First protein to bind to origin recognition complex. (eukaryotes only)

RLFs

Replication licensing factors. Bind after replication activator protein. Some are cytosolic, so only have access to chromosome when nuclear membrane dissolves during mitosis. (eukaryotes only)

pre-RC

Pre-replication complex: Combination of DNA, origin recognition complex, replication activator protein, and replicatino licensing factors. (eukaryotes only)

Cyclin-CDK complex, and effects

Cyclins combine wit cyclin-dependent protein kinases and phosphorylate sites on RAP, RLFs, and ORC. Then RAP and RLFs dissociate and are degraded. (Thus, cyclin-CDKs initiate DNA replication AND prevent formation of another pre-RC.)

Eukaryotic DNA polymerases

α: adds ~20 nucleotides and is replaced by δ and ε.
δ: principal DNA polymerase in eukaryotes. Interacts with PCNA protein (proliferating cell nuclear antigen), which is the eukaryotic equivalent of the "sliding clamp" on the DNA.
ε: Involved in leading strand.
β: Repair enzyme.
γ: DNA replication in mitochondria.

Eukaryotic vs. prokaryotic polymerases

Separate exonucleotic enzymes exist in animal cells (in prokaryotes, the polymerases also act as exonucleases).

Primase activity in eukaryotic cells

Associated with Pol α (addition of RNA primer). Contrasts with primase enzyme in prokaryotes.

FEN-1 and RNase H1

Proteins that degrade RNA primer. Pol δ fills in gaps. DNA ligase seals nicks between fragments.

RPA

Single-stranded binding protein in eukaryotes that protects DNA from degradation.

Histones

Proteins with which eukaryotic DNA is complexed. Histone biosynthesis occurs concurrently with DNA replication.

Prokaryotes vs. Eukaryotes: polymerases

Prokaryotes: 5
Eukaryotes: 5

Prokaryotes vs. Eukaryotes: exonuclease activity

Prokaryotes: polymerases are ALSO exonucleases
Eukaryotes: Not all polymerases are exonucleases (there are separate enzymes to do this)

Prokaryotes vs. Eukaryoes: the origin of replication

Prokaryotes: ONE origin of replication
Eukaryotes: MULTIPLE origins of replication

Prokaryotes vs. Eukaryotes: Okazaki fragments

Prokaryotes: 1000-2000 residues long
Eukaryotes: 150-200 residues long

Prokaryotes vs. Eukaryotes: DNA complex

Prokaryotes: No DNA/protein complex
Eukaryotes: Have histones complexed to DNA

Topoisomerases

Enzymes that relieve torsional strain in coiled DNA (both prokaryotes and eukaryotes). In prokaryotes: DNA gyrase.

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