Nucleic Midterm 2

3′ --> 5′

Original Watson & Crick model required that while one strand would be copied in a 5′ —> 3′ direction, the other has to be copied in ________ direction

5’ —> 3’

Early enzymology work on DNA synthesis found that it could proceed only in a ________ direction

Simultaneously

Radio-labelling experiments showed both strands at a replication fork were being synthesized ___________

Leading Strand, Lagging Strand

Reiji Okazaki suggested that synthesis on one strand ( _________) was continuous while synthesis on the other strand (__________) was discontinuous.

Chases

On leading strand of replication fork, the DNA pol ‘________’ the fork  in 5′ —→ 3′ direction from an exposed 3′ hydroxyl (-OH)

Lagging Strand

needs to wait for upstream DNA 3′-OH to be exposed

Okazaki Fragments

On lagging strand, short bits of new DNA added in are “______________”

Covalently

Eventually, Okazaki fragments are __________ linked together

1000-2000

Prokaryotic Okazaki Fragments are _____-______ nucleotides (nt) long

100-400

Eukaryotic fragments are _____-_____ nt long (fragment length due to processivity)

Many

The lagging strand needs _______ primers

Primase

“_________”, a RNA polymerase makes RNA without a primer (5-10 nt)

De Novo

No DNA polymerases can initiate DNA synthesis ________ (from the beginning)

It is synthesized continuously in the direction of replication fork movement.

Why does the leading strand need only one primer?

How many primers does the lagging strand require?

How many primers does the lagging strand require?

They provide a starting point for DNA polymerase to extend

What is the role of RNA primers in DNA replication?

GTA, an unusually short DNA sequence motif

What DNA sequence helps RNA polymerase bind in E. coli?

RNase H, 5′ exonuclease

RNA primers are removed by _________ and a __________….. (H for hybrid)

DNA Ligase

Nicks are sealed by __________

RNA

RNase H only binds _______ bound to DNA

NAD⁺

E. coli DNA ligase uses _______ as a cofactor…..

Mg²⁺ATP

T4 virus DNA ligase & other phage (& eukaryotic DNA ligases) use __________

DNA Helicase

unwind DNA at the replication fork

Replication Fork

DNA helicases unwind DNA at the ______

Hexameric protein

A protein made of six subunits

Hexameric

DNA helicases are ______ proteins that encircle one DNA strand

Processivity

The ability of an enzyme to perform repeated reactions without falling off its substrate.

Processive

DNA helicases are highly ______, meaning they unwind multiple bases without falling off DNA

ATP

Provides energy for helicases to break base-pair hydrogen bonds and move along DNA

Loaded

DNA helicases are ______ onto DNA to begin unwinding

ATP

DNA helicases use ______ to unwind DNA and move along the strand.

Primase

DNA helicases activate ________

Single Stranded

DNA is _________ after DNA helicase unzips it

Single-Stranded DNA (ssDNA)

can be a hallmark of viral DNA

Single-Stranded DNA Binding proteins (SSB)

ssDNA is stabilized by ________________

Single-Stranded DNA Binding proteins (SSB)

Proteins that bind to and stabilize unwound DNA, keeping it open for replication.

Cooperative Binding

When one bound SSB makes it easier for additional SSBs to bind nearby.

Cooperative

SSBs exhibit ______ binding, where one SSB helps others bind nearby.

Protected

SSBs bind along ssDNA until it is ______ and ready for replication

Phosphodiester

SSBs bind to the DNA ______ backbone and stack with nucleotide bases.

Hydrogen

Unlike normal DNA base pairing, SSBs do not form ______ bonds with ssDNA

Viral

Single-stranded DNA can be a hallmark of ______ DNA.

Open

SSBs hold unwound DNA ______ for DNA replication.

Positively

DNA becomes ___________ supercoiled due to replication fork moving

Positive supercoiling

Overwinding of DNA that builds up ahead of the moving replication fork.

10

DNA linking number needs to be reduced by cutting DNA approximately every ______ base pairs.

Topoisomerases

Enzymes that relieve DNA supercoiling by cutting and rejoining DNA.

Unreplicated

Topoisomerases act on ______ DNA ahead of the replication fork.

DNA topoisomerase II

An enzyme that cuts both DNA strands, passes another DNA segment through the break, and rejoins the strands.

Both

Topoisomerase II breaks ______ DNA strands and holds onto both strands.

Overwinding creates tension that makes further DNA unwinding difficult.

Why is supercoiling a problem?

Circular

Supercoiling is a major problem in ______ DNA because it cannot freely rotate to release twisting.

DNA polymerase (III)

extends DNA

DNA polymerase I

replaces gaps where RNase H removed RNA primer and an exonuclease digested the last RNA-DNA link

Replisome

a group of proteins and enzymes that work together at the replication fork to replicate DNA.

DNA polymerase III

Extends RNA primers by adding DNA nucleotides in the 5′ to 3′ direction.

Multiple

Both prokaryotes & eukaryotes have ______ DNA polymerases

DNA polymerase I

not processive (20-100nt/binding), removes upstream RNA from Okazaki fragment, fills in gap left by RNase H, own 5′ exonuclease activity

DNA polymerase III

highly processive since it replicates 4.6 Mb E. coli genome from only 2 replication forks

5

Prokaryotes have ____ DNA Pol isoforms, 2 “hi-fi” for replication, 3 for repair only

Isoforms

Prokaryote DNA Polymerase ________ distinguished not only by specific function, but also enzyme kinetics, subunit composition, & abundance

3

Eukaryotes have many DNA Pol isoforms, ____ for replication (can interchange)

Primase

DNA Pol α/primase H starts off with ______ activity then DNA pol activity (but has low processivity)

DNA polymerase ε

Primarily synthesizes the leading strand

DNA polymerase δ

Primarily synthesizes the lagging strand

DNA polymerase α/primase

Initiates DNA replication by making an RNA primer and extending it with a short stretch of DNA.

Polymerase switching

The transition from DNA polymerase α to the more processive DNA polymerases ε and δ.

Active Sites

________ and catalytic mechanisms of polymerases are highly conserved

Evolutionary Lineage

Polymerases are grouped according to _____________ of rest of the protein

6

DNA Polymerases are in ____ main groups based on their similarities and functions

Evolutionary Divergence

Polymerases are more similar within groups than within organisms, suggesting early __________________

Processivity

High ____________ at replication fork needed for fast chromosome duplication

Sliding Clamps

bind DNA Pol, keeps it from diffusing away and on track

Sliding Clamps

encircles DNA, binds to, vastly improves processivity of  DNA Pol

Sliding clamp-DNA Pol complex

dissociates when it hits double-stranded DNA

dsDNA

DNA Pol recognizes dsDNA, active site has low affinity for _________ (as opposed to high affinity for RNA primer:DNA template junction)

RNA primer–DNA template

DNA polymerase has high affinity for the ______ junction

Double-Stranded

DNA polymerase has low affinity for ______ DNA (dsDNA).

Conformational

When DNA polymerase encounters dsDNA, it undergoes a ______ change.

Polymerase

After the conformational change, the sliding clamp has low affinity for DNA ______.

Encircling

When DNA polymerase detaches, the sliding clamp remains ______ the DNA.

PCNA

The eukaryotic sliding clamp that holds DNA polymerase on DNA and also participates in nucleosome assembly.

Nucleosome

PCNA remains on DNA after DNA polymerase detaches and helps with ______ assembly

Sliding clamp loader

A protein complex that uses ATP to open sliding clamps, load them onto DNA, and help remove them.

ATP

Sliding clamp loaders use ______ to open the sliding clamp ring.

Subunits

Sliding clamps are made of tightly associated protein ______.

Sliding clamp loading

The process of opening a clamp, placing it around DNA, and allowing it to close.

Assembly

Sliding clamp loaders help remove clamps after DNA replication and nucleosome ______ are complete.

Binding

DNA polymerase and sliding clamp loaders compete for the same ______ site on the clamp.

Sliding clamp roles beyond replication

Sliding clamps also help recruit proteins involved in DNA repair and nucleosome assembly.

On and off

Sliding clamp loaders help put sliding clamps ______ DNA and remove them when needed.

Removal allows DNA to pair appropriately and permits other DNA-processing activities

Why must sliding clamps be removed?

Slide Clamp Loading

(a) Slide clamp loader protein (‘З-like’ protein) senses primer:template junction

(b) binding of ATP, conformational change

(c) binds to protein:protein interaction domain of sliding clamp protein, breaks hexameric ring

(d) recognizes p:t junction, slips ring onto DNA strand

(e) DNA binding causes ATP hydrolysis by altering conformational shape, causing dissociation, DNA is left encircled by clamp protein

Replication Factor C

Slide clamp loader protein is γ-complex in E. coli, ____________ (RF-C) in eukaryotes

Conformational Change

Slide clamp loader protein uses ATP and ____________ to load clamp protein

Difficulty, multiple

Cells must minimize time that DNA is single-stranded (ssDNA) due to _______ of repair ——> _________ DNA Pol are active at replication fork

Physically Linking

E. coli DNA replication can be coordinated by ________ enzymes (DNA Pol III holoenzyme)

Holoenzyme

1 core enzyme activity + accessory proteins to enhance core enzyme function

Tau (τ)-protein

links clamp loader with two DNA Pol III subunits (1 for leading + 2 for lagging strand synthesis)

DNA Pol III holoenzyme

is a complex of DNA Pol, clamp loader, & sliding clamp