Lecture 2 DNA Replication

What are the three stages of DNA Replication?

Initiation, Elongation, and Termination

What does it mean that DNA replication is "semiconservative"?

Each daughter cell contains one parental (old) DNA strand and one newly synthesized strand

What does it mean that DNA replication is "bidirectional"?

Two replication forks move in opposite directions away from the origin, each copying one parental strand

What is the origin of replication in E. coli called?

OriC

What protein recognizes and binds OriC to initiate replication?

DnaA (the initiator protein)

What is DnaB?

A DNA helicase enzyme that separates the parental DNA strands to open the replication bubble

What enzyme unwinds DNA ahead of the replication fork by relieving torsional strain, and is a bacterial topoisomerase II?

DNA gyrase

What do single-stranded binding proteins (SSBPs/SSBs) do?

Bind ssDNA to remove secondary structure, protect it from digestion, and keep the strands apart so they don't reanneal

What enzyme lays down the RNA primer needed before DNA polymerase can begin synthesis?

Primase (a type of RNA polymerase)

Why can't DNA polymerase initiate replication on its own?

It cannot bind or begin synthesis without an existing RNA primer providing a free 3'-OH group

In what direction is the parental DNA template read by DNA polymerase?

3' to 5'

In what direction are new nucleotides added to the growing DNA strand?

5' to 3'

Which E. coli DNA polymerase is the main polymerizing enzyme responsible for the bulk of elongation?

DNA Polymerase III

Which E. coli DNA polymerase functions in proofreading, repair, and RNA primer removal?

DNA Polymerase I

Which E. coli DNA polymerase functions mainly in DNA repair (not the main replicase)?

DNA Polymerase II

What cofactor is required for the nucleotide addition reaction catalyzed by DNA polymerase?

Mg2+

What does "processivity" mean for a DNA polymerase?

The ability to continuously synthesize DNA without dissociating from the template

What is the β2-sliding clamp?

A subunit of DNA Pol III that tethers the polymerase to DNA, ensuring processivity

What loads the β2-sliding clamp onto the RNA primer-template junction?

The clamp-loader complex

What is the Replisome?

The entire large complex of proteins that carries out DNA replication, forming the replication fork for both leading and lagging strands

What is the leading strand and how is it synthesized?

The strand synthesized continuously toward the replication fork (away from the origin), requiring only one primer

What is the lagging strand and how is it synthesized?

The strand synthesized discontinuously, away from the replication fork (toward the origin), in short pieces requiring many primers

What are Okazaki fragments?

Short, discontinuous stretches of DNA synthesized on the lagging strand

What enzyme removes the RNA primers between Okazaki fragments in prokaryotes?

RNase H

What enzyme fills in the gaps left after RNA primer removal between Okazaki fragments?

DNA Polymerase I

What enzyme joins the ends of DNA fragments together to form a continuous phosphodiester backbone?

DNA ligase

What is the "Trombone Model" of DNA synthesis?

A model describing how the DNA Pol III complex on the lagging strand loops the template so it can synthesize in the same direction as the leading strand, then jumps to the next RNA primer after finishing each Okazaki fragment

What is catenation in the context of DNA replication termination?

The linking together of two circular daughter DNA molecules after replication, which must be resolved before the cells can divide

What resolves catenated daughter chromosomes in E. coli?

Type II topoisomerases (gyrases), which break one strand, separate the catenated chromosomes, and reseal the DNA

What are Ter sites?

DNA sequences where replication forks are physically halted during termination

What protein binds Ter sites to stop the replisome/helicase from advancing?

Tus (Terminus Utilization Substance)

Name three features shared by prokaryotic and eukaryotic DNA replication.

Initiation requires an RNA primer, elongation proceeds 5' to 3', and replication is continuous on the leading strand but discontinuous on the lagging strand

How do bacterial and eukaryotic chromosomes differ in shape?

Bacterial chromosomes are circular; eukaryotic chromosomes are linear

How many origins of replication does a eukaryotic chromosome typically use, compared to a bacterial chromosome?

Thousands of origins in eukaryotes, versus a single origin (OriC) in bacteria

Which eukaryotic DNA polymerases are primarily responsible for leading and lagging strand elongation?

DNA Polymerase ε (epsilon, leading strand) and DNA Polymerase δ (delta, lagging strand)

Which eukaryotic DNA polymerase lays down the initial RNA primer along with its associated primase?

DNA Polymerase α (alpha)

What is the Origin Recognition Complex (ORC)?

A protein complex that binds origins of replication across eukaryotic chromosomes to initiate pre-replication complex assembly

What two proteins are recruited by ORC to help assemble the pre-replicative complex (pre-RC)?

Cdc6 and Cdt1

What is the eukaryotic replicative helicase loaded during pre-RC formation?

The Mcm2-7 helicase complex

During what phase of the cell cycle does pre-RC (origin licensing) assembly occur, and why?

G1 phase, because Cdk activity is low at that time (CDKs inhibit pre-RC assembly)

What two kinases activate ("fire") licensed origins during S phase?

CDKs (cyclin-dependent kinases) and DDK (Dbf4-dependent kinase)

What prevents a new pre-RC from forming at an origin until the next G1 phase?

High Cdk activity during S, G2, and M phases, which disassembles the pre-RC after firing

What is PCNA?

Proliferating Cell Nuclear Antigen — a ring-shaped sliding clamp that provides processivity for eukaryotic DNA polymerases; also used clinically as a marker of proliferating (cancer) cells

What is the eukaryotic clamp loader called?

RFC (Replication Factor C)

What is the eukaryotic single-stranded DNA binding protein equivalent to bacterial SSB?

RPA (Replication Protein A)

What two enzymes remove RNA primers between Okazaki fragments in eukaryotes?

RNase I and FEN1 (Flap Endonuclease 1)

What are the four phases of the eukaryotic cell cycle (plus the resting phase)?

G1 (growth), S (DNA synthesis), G2 (preparation for mitosis), M (mitosis/division), and G0 (quiescence)

During which phase of the cell cycle does DNA replication occur?

S phase

What are cyclins and cyclin-dependent kinases (Cdks)?

Regulatory proteins that control progression through the cell cycle; cyclins have no activity alone but activate Cdks when bound, driving cell cycle transitions

What is the Restriction Point (R)?

The point near the end of G1 phase after which a cell no longer needs growth factors to complete the cell cycle

What are the two major cell cycle checkpoints?

The G1–S checkpoint and the G2–M checkpoint

What is the "End Replication Problem"?

The inability to fully replicate the very 3' end of a linear chromosome's lagging strand because there is no way to prime and fill the gap after the last RNA primer is removed (no 3'-OH available)

Why isn't the End Replication Problem an issue for circular (bacterial) chromosomes?

Because there is no chromosome "end" — the excised primer gap can always be filled using an adjacent 3'-OH

What are telomeres?

Repetitive DNA sequences (TTAGGG in humans) that cap the ends of linear chromosomes and protect them from shortening and degradation

What enzyme extends telomeres, and what is unique about it?

Telomerase — it is a specialized reverse transcriptase that carries its own RNA template to add telomeric repeats to the chromosome end

What is TERT?

Telomerase Reverse Transcriptase — the catalytic protein subunit of telomerase that uses the telomerase RNA template to synthesize telomeric DNA

After TERT extends the leading strand overhang, how is the complementary (lagging) strand of the telomere completed?

A specialized DNA polymerase fills in complementary bases, and DNA ligase seals the DNA together

What happens to telomeres each time a normal somatic cell divides?

They shorten (roughly 25–200 base pairs lost per division) because telomerase is normally inactive

What happens to a cell when its telomeres become critically short?

The cell can no longer replicate and typically dies via apoptosis (or enters senescence)

In which normal human cell types is telomerase activity chronically high?

Undifferentiated embryonic stem cells, male germ cells, and activated lymphocytes

Why are most cancer cells considered "immortal"?

Because they overexpress telomerase, maintaining telomere length and allowing indefinite proliferation (~85% of cancers show elevated telomerase activity)

What is Werner Syndrome and its genetic cause?

An autosomal recessive "adult progeria" disease of premature aging caused by mutations in the WRN gene (a DNA helicase), impairing DNA repair and telomere maintenance

What do IPF and DC stand for in the context of telomere-shortening syndromes?

Idiopathic Pulmonary Fibrosis and Dyskeratosis Congenita

What DNA structural feature explains why DNA is more resistant to base-induced hydrolysis than RNA?

The absence of a 2'-hydroxyl group in deoxyribose

What conformation of DNA do poxviruses (e.g., variola/smallpox) target to hijack host replication machinery?

Z-DNA form

What is a DNA gyrase, functionally classified?

A bacterial topoisomerase II

Through what mechanism must the two DNA strands physically separate for replication to occur?

The action of helicase enzymes

What is Nucleosomes' main function?

Compacting DNA (by wrapping it around histone proteins to form chromatin)

What group is missing at the very end of a linear chromosome's lagging strand that causes the End Replication Problem?

A 3'-OH group