DNA Replication

DNA

linear molecule with defined double helix structure composed of nucleotide bases (A,T,C,G)

2-deoxy-D-ribose

5 carbon (pentose) sugar component of DNA

nucleotide

monomer of nucleic acids made up of a 5-carbon (pentose) sugar, one or more phosphate group, and a nitrogenous base

nucleoside

A nitrogenous base + pentose sugar

(no phosphate group)

nucleobase

Pyrimidines (C, T, U) and purines (A, G)

T/F: RNA is less stable than DNA

True. RNA has a highly reactive OH group (can be degraded) at the 2' carbon whereas DNA only has a hydrogen there

Where are nucleotides added on a growing strand of DNA or RNA?

3' OH group

growing end

replication fork

A Y-shaped region on a replicating DNA molecule where new strands are growing.

alpha phosphorous

Elongation in DNA synthesis requires repeated nucleophilic attacks here of the incoming 5' dNTP

What is released in the reaction that occurs when adding a new base pair in the elongation phase of DNA?

Inorganic phosphate

okazaki fragment

Short pieces of DNA fragments that are later joined together to make the lagging strand of DNA

T/F: The lagging strand is looped around during replication

True. This is so that DNA polymerase III can synthesize both strands at the same time

leading strand of DNA

synthesized continuously, but a free 3'OH is missing at the origin without an RNA primer

lagging strand of DNA

Daughter strand of DNA synthesized in short segments that are later joined. Synthesis of the lagging strand always moves away from the replication fork but occurs in a discontinuous manner, and lags behind synthesis of the leading strand

Why is discontinuous DNA synthesis required in order to copy the lagging strand?

The strand is oriented such that the 5' is in the direction of replication, but the growing end is the 3' end

Fidelity

ability of DNA polymerase to avoid or to correct errors during synthesis of DNA strands

Processivity

ability of DNA polymerase to carry out a continuous stretch of DNA synthesis without dissociation from a template

RNA primase

An enzyme that creates an RNA primer complementary sequence for initiation of DNA replication

T/F: Both DNA and RNA polymerases require a free 3'-OH group to start synthesis?

False. Only needed by DNA polymerase, not RNA polymerase

T/F: Both the leading and lagging strands require primase to generate an RNA primer

True

origin

Defined region in DNA (not random) where replication begins (initiation)

Single stranded binding proteins

bind to unwound DNA to stabilize single strands

Topoisomerase Type I

breaks ONE of the two DNA strands

Topoisomerase Type II

breaks BOTH DNA strands

DNA gryrase

Type II topoisomerase enzyme that removes supercoils in DNA during initiation of replication, relieving strain on DNA. It also introduces supercoils in DNA. If it removes the twist, it must add it back later to reseal the DNA.

How many DNA gyrases are needed for bidirectional DNA replication?

2

Helicase

breaks the hydrogen bonds between nucleotide base pairs in double-stranded DNA

T/F: DNA helicase requires ATP

True. Energy is needed to break the hydrogen bonds

Supercoiling

Process of coiling a coil. Controlled by topoisomerase enzymes. Supercoiling forms a compact form of DNA, which is how it can fit in the small nucleoid

Phosphodiester bond

the type of bond that links the nucleotides in DNA or RNA. Joins the phosphate group (5') of one nucleotide to the hydroxyl group (3') on the sugar of another nucleotide. Bonds form via a condensation reaction

Nucleosome

Bead-like structure in eukaryotic chromatin, composed of a short length of DNA wrapped around a core of histone proteins.

How does DNA wrap so tightly in a histone?

DNA is negatively charged and is highly attracted by the positively charged histone core proteins

Chromatin

DNA complexed with protein (histones/nucleosomes) in eukaryotic cells

Structure of human chromosomes in which DNA is tightly packed

Core histone

Positively charged octamer protein that DNA wraps around, forming a nucleosome

H2A, H2B, H3, H4

What amino acids enrich histones?

Lys and Arg

Linker histone

Histone H1

DNA not associated with core histones

Nucleoid

A non-membrane-bounded region in a prokaryotic cell where the DNA is concentrated in a circular chromosome

Site of prokaryotic DNA replication

DNA ligase

enzyme used to close a nick in DNA

What kind of bond does DNA ligase generate?

Phosphodiester

Catenane

DNA molecules that are interlocked but not covalently attached

Result of DNA replication ending, where two double stranded copies of the bacterial chromosome are interlocked.

T/F: Topoisomerase I can be used to separate a catenane

False. A catenane has two double stranded copies of DNA, needing Topoisomerase II to break both strands

Chargaff's rules

no matter the organism studied, chemical analysis of DNA always showed A=T and G=C, or purines = pyrimidines

Purines

Adenine and Guanine

Pyrimidines

cytosine, thymine, uracil

Types of bonds between purines and pyrimidines

2 hydrogen bonds between A and T

3 hydrogen bonds between G and C

The bonds between which nucleobases are hardest to separate? Why?

GC rich regions are hard to separate due to 3 hydrogen bonds (as opposed to 2 in AT rich regions, which facilitate separation of stands for replication).

What direction is DNA replication?

5' to 3'

What direction is RNA transcription?

5' to 3'

Semiconservative DNA replication

During DNA replication in both prokaryotes and eukaryotes, two new DNA molecules are made, each with a new strand and an old strand, generated during S phase of cell cycle

DNA polymerase I

Removes RNA primers made by primase using its 5'-3' exonuclease activity, and fills gap between Okazaki fragments on lagging strand only. Low processivity

What is the proofreading activity of DNA polymerase I?

5'-3' exonuclease (removal of RNA primers, and used in DNA repair)

and

3'-5' exonuclease

DNA polymerase III

The principle replication polymerase. Makes DNA on leading AND lagging strand. Makes Okazaki fragments. High processivity.

What is the proofreading activity of DNA polymerase III?

3'-5' exonuclease

T/F: Exonuclease activity can be coupled to polymerization

True. Nucleotides can be removed from the 5' terminus by DNA pol I which then also replaces the bases

Ter sequences

Regions that serve as the binding site for the Tus protein to end DNA replication

Tus protein

Terminus utilization substance

Binds to Ter sequences on DNA to terminate replication

T/F: Each fork of replication requires its own Tus protein

True. The clockwise fork has its own ter sequences and Tus protein, and the counter-clockwise fork has its own too.

T/F: Incorrect bases can be rejected before the phosphodiester bond is formed

True. While it is possible for an incorrect nucleotide to hydrogen bond with a base in the template, it generally does not fit into the active site. This is the rejection before a true bond is formed in synthesis

What is the error rate for DNA polymerase?

1 error every 10^6 bases

Location of DNA synthesis in eukaryotes

Cell nucleus

When does DNA synthesis occur in eukaryotes?

S phase of cell cycle

T/F: There is a single origin of replication in eukaryotic DNA

False. There are multiple origins in eukaryotes, but only one in prokaryotes. This explains how the slow activity of DNA polymerase in eukaryotes is compensated for.

What are similarities between prokaryotic and eukaryotic DNA replication?

1. Need for primase

2. Leading and lagging strand

3. DNA polymerase enzymes have different rates of processivity