BIOL 330 Exam 1 Lectures 3-6

What are viruses?

Genetic material encased in a protein coat called a capsid that can infect host cells and replicate.

When the polymerase adds an incorrect nucleotide, the newly synthesized DNA strand does what?

Transiently unpairs from the template strand, and its 3’ end moves into the editing site (E) to allow the incorrect nucleotide to be removed

Characteristics of bacterial chromosomes?

• Circular, double-stranded DNA compacted into nucleoid

• Their DNA is associated with HU and H-NS DNA-binding proteins

• Readily replicated and transcribed

What activity provides DNA pol III the ability to proofread?

The 3' to 5' exonuclease activity allows DNA pol III to remove incorrectly paired nucleotides

Telomeres and Telomerase

Telomeres are repetitive nucleotide sequences at the ends of chromosomes that protect them from degradation

Telomerase is an enzyme that extends these telomeres, preventing loss of genetic information during DNA replication

Characteristics of viral chromosomes?

• Linear or circular genetic material, which can be DNA or RNA

• Often single-stranded or double-stranded

• Encased in a protein coat and may have an envelope

• Carries the information necessary to hijack host cell machinery for replication, transcription, and translation to produce new viral particles

Bacterial DNA is highly folded into a ?

Series of twisted loops

Topoisomerases

• Responsible for adding and removing turns in the coil

• Wind or unwind helix before resealing

What is chromatin?

A highly organized condensed structure made up of DNA, RNA, and proteins that forms the chromosomes in the nucleus of a cell

What is a histone?

Prevent DNA from becoming tangled and protect it from DNA damage and contain large amounts of lysine and arginine

What makes electrostatic bonding to negatively charged phosphate possible?

The positively charged amino acids in histones

H1 histone

Stabilize nucleosome structure and higher-order chromatin architecture by pulling nucleosomes together into a regular repeating array

Neutralizing their positive charges would have which effect on the histone proteins?

They would bind less tightly to DNA

The nucleosome core particle can be released from chromatin by?

Digestion of the linker DNA with a nuclease

Loosely packed chromatin is called

Euchromatin

During interphase a few regions of chromatin (centromeres and telomeres) are ?

Highly condensed into heterochromatin

dense packing of the heterochromatin makes it difficult for?

The cell to express genetic information coded in these regions

What are the three characteristics of Euchromatin?

It becomes less tightly compacted after cell division

It is accessible to enzymes needed for gene expression

It includes DNA primarily found in expressed genes

Characteristics of Heterochromatin?

• Genetically inactive

• Replicates later in S phase than euchromatin

Telomere vs Centromere

Telomeres are protective caps at the ends of chromosomes that prevent degradation

Centromeres are regions that link sister chromatids and are crucial for proper chromosome segregation during cell division

To allow replication and gene expression, chromatin must?

• Relax compact structure

• Expose regions of DNA to regulatory proteins

• Have a reversal mechanism for inactivity

What is the purpose of histone tails?

Provide sites for chemical modifications that can include methylation, acetylation, phosphorylation, and ubiquitination, influencing the activity of nearby genes

What do histone deacetylase (HDAC) enzymes do?

Remove acetyl groups from histone tails, leading to chromatin condensation and reduced gene expression

What does acetylation do?

Acetylation is a process where acetyl groups are added to histone tails, leading to chromatin relaxation and increased gene expression

• positive correlation with gene activity

Open configuration means?

DNA is unmethylated and histones are acetylated so genes can be transcribed

Closed configuration means?

DNA is methylated and histones are deacetylated so genes cannot be transcribed

Methylation is?

The addition of methyl groups to arginine and lysine, leading to gene silencing and reduced transcription

• repress or promote gene regulation

Phosphorylation is?

The addition of phosphate groups to hydroxyl groups of amino acids such as serine (S), threonine (T), histidine (H), tyrosine (Y)

• positive correlation with gene activity

Bacterial genomes consist of?

Mostly unique DNA sequences coding for proteins

Eukaryotic genomes contain a mixture of?

Both unique and repetitive DNA sequences

rDNA is?

A type of repetitive DNA that codes for ribosomal RNA, essential for ribosome production and function

Satellite DNA (satDNA)

A type of repetitive DNA that consists of short, tandemly repeated sequences, often found in the heterochromatin regions of chromosomes

The vast majority of Eukaryotic genome does not?

Encode functional genes or proteins

Pseudogenes

Duplicated copies of genes that have undergone considerable mutation and share some homology to the original gene

• not capable of coding for a protein

• are NOT transcribed

Common defects of pseudogenes?

• Missing promoters

• Missing start codon

• Frameshift

• Premature stop codon

• Missing introns

• Partial deletion

Conservative model

A hypothesis about DNA replication proposing that one strand of the parent DNA is conserved in each daughter molecule

• One parental DNA molecule and 3 new molecules

Semiconservative model

A hypothesis about DNA replication stating the two strands of the parental molecule separate, and each function as a template for synthesis of a new, complementary strand

• Two DNA molecules that are half parental str4and DNA and half new strand and two DNA molecules that are new

Dispersive model

A hypothesis about DNA replication suggesting that each strand of both daughter molecules contains a mixture of old and newly synthesized DNA

• four DNA molecules that contain both parental and new DNA

DNA is reproduced by?

Semiconservative replication

Centrifugation in a cesium chloride (CsCl) gradient allows the separation of heavy and light DNA, how?

The DNA will migrate to the region where its density matches that of the salt surround it

In Meselson and Stahl’s experiment proving semiconservative DNA

replication, they started with bacteria grown in a heavy isotope of

nitrogen and then switched them to a light isotope. They then

observed the DNA density after one and two rounds of replication.

What was the result after two rounds of replication?

Equal amounts of light and hybrid DNA

DNA replication begins at the?

ORI (origin of replication) made up of mostly A/T base pairs

• only one ORI

Replication fork

The area where the DNA double helix unwinds to allow for the synthesis of new strands during DNA replication

Replication is bidirectional, which means?

There are two replication forks

Replicon is?

A segment of DNA that replicates from a single origin of replication, encompassing one or more genes

DNA runs?

in anti-parallel strands, meaning one strand runs 5' to 3' and the other runs 3' to 5'

DNA adds one nucleotide at a time to the?

3’ end of the growing strand

When nucleotide is added, what happens?

Two terminal phosphates cleaved off, providing newly exposed 3’ -OH

DNA polymerase

An enzyme responsible for synthesizing new DNA strands by adding nucleotides to the 3’ end of a growing strand during DNA replication

dNMP vs dNTP

dNMP: deoxyribonucleotide monophosphate - one phosphate group

dNTP: deoxyribonucleotide triphosphate - three phosphate groups

DNA replication occurs by adding ______.

dNTPs to the 3’ of the daughter strand

Template strand vs daughter strand?

Template strand is used for copying and daughter strand is where you add nucleotides

DNA polymerase III

An enzyme involved in DNA replication that synthesizes new DNA strands by adding dNTPs to the growing daughter strand

• Builds DNA

DNA polymerase I and II

DNA pol I:

• removes primers on lagging strand

• synthesize short stretches of DNA (gap filling)

DNA pol II:

• Proofreading during DNA replication and repair

DNA Pol I, II, and III can and cannot?

• Can elongate (5’-3) existing DNA strand (primer) with 3’ -OH

• Cannot initiate DNA synthesis

Exonuclease activity 3’ to 5’

All three DNA Pol have this:

• Proofread newly synthesized DNA

• Remove/replace incorrect nucleotides

Exonuclease activity 5’ to 3’

• Involved in removing RNA primers during DNA replication

• Excises primers—fills in gaps left behind

Holoenzyme consists of?

Apoenzyme (protein portion) + cofactors

Holoenzyme of DNA polymerase III consists of three assemblies, they are?

the core enzyme, a gamma clamp-loading complex, and a beta sliding clamp

The pol III core does what?

Elongates polynucleotides chain and proofread

The beta sliding clamp does what?

Holds the DNA polymerase in place on the DNA strand during replication, preventing it from dissociating

The gamma clamp-loading complex does what?

It facilitates the placement of the beta sliding clamp onto the DNA, allowing DNA polymerase to efficiently initiate replication

Sliding DNA clamp vs sliding DNA clamp loader?

SDC: keeps DNA polymerase attached to the template, allowing the enzyme to move along without falling off as it synthesis new DNA

SDCL: Uses the energy of ATP hydrolysis to load the sliding clamp and lock it onto DNA

What are the seven key issues that must be resolved during DNA replication?

1. Unwinding of helix

2. Reduce increased coiling generated during unwinding

3. Synthesis of primer for initiation

4. Discontinuous synthesis of second strand

5. Removal of the RNA primers

6. Joining of gap-filling DNA to adjacent strand

7. Proofreading

Helicases are?

Enzymes that untwist the double helix at the replication forks

Initiator proteins

• Binds to ORI causing conformation change

• Causes helix to destabilize and opens up

• Exposes ssDNA

DNA helicase

A type of helicase enzyme that unwinds the DNA double helix at the replication fork, allowing the replication process to proceed

Single-stranded binding proteins (SSBPs)

They are proteins that bind to and stabilize single-stranded DNA during replication, keeping the strands open so helicase can unwind the DNA without it snapping back, and they act on both the leading and lagging strands.

What causes the unwinding of helix?

• Helicases

• Single-stranded binding proteins

DNA gyrase

Functions both as a restriction endonuclease and as a ligase by cleaving and rejoining supercoiled DNA ends to facilitate replication

Restriction endonuclease?

An enzyme that cuts DNA at specific recognition sites, often used in molecular cloning

Ligase

• Catalyzes formation of phosphodiester bonds

• Seals nicks and joins Okazaki fragments together

Topoisomerase I vs II

Topoisomerase I cuts one strand of DNA to relieve supercoiling, while Topoisomerase II cuts both strands to introduce negative supercoils

Primase

An enzyme that synthesizes short RNA primers during DNA replication (joins together two ribonucleotides) in the 5’ to 3’

Primase can start a new polynucleotide chain by joining together two nucleoside triphosphates without the need for?

A base-paired 3’ end as a starting point

RNA primer

A short sequence of RNA that provides a free 3' hydroxyl group for DNA polymerase to initiate DNA synthesis

RNA primer provides?

Free 3’ -OH required by DNA polymerase III for elongation

RNA primers are synthesized by an _____ _____ called ______ which uses DNA strand as a template

RNA primers are synthesized by an RNA polymerase called primase, which uses a DNA strand as a template.

Pol S (delta) vs Pol E (epsilon)

Pol S synthesizes lagging strand

Pol R synthesizes leading strand

Pol A (alpha)

RNA/DNA primers, initiation of DNA synthesis

Translesion DNA synthesis

Facilitate DNA replication (and thus cell division) by efficiently bypassing various DNA lesions that stall the replication machinery

Error-prone DNA polymerases

Can synthesize DNA across damaged DNAs but at the cost of introducing mutations

In prokaryotes, DNA replication occurs in the? And is not restricted to?

cytoplasm; the nucleus

How is eukaryotic DNA replication more complex?

• More DNA than prokaryotic cells

• DNA complexed with nucleosomes

• Cell cycles

• Linear chromosomes

Centromere vs Telomeres

Centromere: Attachment point for spindle microtubules

Telomeres: Tips of a linear chromosome

What is a DNA-dependent RNA polymerase?

A primase

Telomerase reads ______ as a template and synthesizes _____ as a product

RNA; DNA