Biochem 285 Exam 2

What are the three components of DNA?

sugar, phosphate group, nitrogenous base

Purines

Adenine and Guanine. 2 structures.

Pyrimidines

Cytosine and Thymine. 1 structure.

Genome

all of an organism's genetic material

Gene

sequence of DNA that codes for a protein and thus determines a trait

Gene isoforms

mRNAs that are produced from the same locus but are different in their transcription start sites (TSSs), protein coding DNA sequences (CDSs) and/or untranslated regions (UTRs), potentially altering gene function.

Chromatin

Linear DNA wound around histones and non-histone proteins. Histones alter chromatin structure.

Euchromatin

loosely packed chromatin. histones are wound around DNA but short stretches of DNA are exposed to other proteins

Heterochromatin

condensed chromatin. Genes not expressed.

Nucleosome

Double stranded DNA wraps around histones ~1.5 times to make a nucleosome. A histone is made up of 8 protein subunits, each of which have N-terminal "Tails" that can be chemically altered by enzymes. The modifications placed on the histone tails help to determine how strong of an association the histone will have with DNA, and thus tighten or relax chromatin structure

What is the protein structure of histone

Histone is an octomer

Acetylation / histone acetyltransferase (HAT)

Adds Acetyl group to Lysine, removes amino group.

Makes charge neutral instead of positive

This weakens the bonds and makes chromatin loose, allowing gene expression.

Histone deacetylases (HDACs)

remove acetyl groups, leading to chromatin condensation and no gene expression

Phosphorylation / Kinase

enzyme that replaces a hydroxyl group with a phosphate group: leads to loosely packed chromatin (due to phosphate having a negative charge like DNA) leading to gene expression. Occurs at the N-terminal tails of histone proteins

Phosphatase

removes a phosphate group from a molecule: leads to tightly packed chromatin

Histone Methylation / Histone methyltransferase (HMT)

methylates lysine and arginine in the histone tails, inhibits gene expression. Acetylated (Ac) chromatin is "immune" to trimethylation by HMT. Occurs at the N-terminal tails of histone proteins.

histone demethylases (HDMTs)

removes methyl groups from histones, activates gene expression.

methylation and acetylation

Methylation competes with acetylation as a methylated positively charged amino acid cannot be acetylated. D. methylation influences histone-histone interactions

DNA Methylation of CpG islands.

CpG islands is where methylation occurs. represses transcription. Protects DNA. Hypermethylation of promoter prevents tumor suppressor gene expression

origin of replication

a particular sequence in a genome at which replication is initiated. Firing of ORIs are linked to distinct stages in the cell cycle

What way is DNA replicated and why?

5' to 3'. During DNA replication, the new (incoming) nucleotide is always added to the 3' carbon's hydroxyl group, meaning the DNA is always replicated in the 5' to 3' direction.

Helicase

Binds DNA at ORI and separates strands without ATP

Primase.

synthesizes RNA primer

DNAP alpha

grows DNA primer by adding bases. Does this on lagging strand

Sliding clamp (PCNA)

loads DNAP in replication fork

DNAP epsilon

adds bases on the leading strand

DNAP delta

adds bases on the lagging strand

DNA ligase

seals nicks in DNA

Okizaki fragments

the fragments formed by the discontinued replication on the lagging strand

Topisomerase

A protein that functions in DNA replication, helping to relieve strain in the double helix ahead of the replication fork. Keeps Helicase binded to DNA

RFC

sliding clap (PCNA) loader

Telomeres

a sequence (DNA nucleotides) at the end of chromosomes that shortens with each replication acting as a biological clock. They are protected by shelterin cap to prevent them from looking like double strand breaks.

Telomerase

An enzyme that extends the 3' end of a DNA parent strand by adding telomers so that a complementary primer can be added in the 5' to 3' direction.

Taq polymerase

A DNA synthesis enzyme that can withstand the high temperatures of PCR

Cloning

Moving DNA fragments into a plasmid to express a specific gene

1. Obtain DNA from PCR

2. Cut DNA fragment using restriction digest

3. Paste DNA in vector using ligase

restriction enzymes / nucleases

Enzyme that cuts phosphodiester bond in DNA at a specific sequence of nucleotides. Isolated from bacteria

agarose gel electrophoresis

Used for size separation of PCR products (smaller molecules travel further); compared against a DNA ladder. DNA moves towards the positive electrode. Different DNA structures with the same number of base pairs move different amounts on the gel so linear DNA is used.

RT-qPCR

A combined process of reverse transcriptase and quantitative PCR in which cellular mRNA is amplified by using RT-PCR to generate cDNA, followed by measuring the relative amount of amplified DNA with qPCR. The amount of cDNA can then be compared against the original samples of mRNA.

Mis-match repair (MMR)

Fixes a point mutation right after DNA synthesis. MutS, MutL, UvrD, Exol, DNA polymerase repairs, Ligase seals nick

MutS

Scans for a mismatch in MMR

MutL

Determines old vs new strand in MMR through methylation patterns.

UvrD

helicase in MMR, unwinds DNA

Exol

exonuclease in MMR, cuts out DNA from the nick

MMR in prokaryotes

uses methylation to determine old vs new strand

Depurination

the loss of a purine (G or A) base from a nucleotide

Deamination

Removal of an Amine group from a C, A , or G base.

C to Uracil

A to hypoxanthine

G to xanthine

Base Excision Repair (BER)

Repair pathway that repairs a single base damaged by deamination or depurination. DNA Glycosylase, AP endonuclease, DNA Polymerase adds correct base, Ligase seals the bond

DNA glycosylase

In BER flips damaged base out and removes base from the phosphate backbone.

AP endonuclease

Recognizes the AP site and removes the phosphate backbone.

pyrimidine dimers

covalent bonds between pyrimidies that form from radiation and causes DNA to not function properly. Repaired by NER

Nucleotide Excision Repair (NER)

a DNA repair system in which several nucleotides in the damaged strand are removed from the DNA to fix pyrimidine dimers. 2 pathways. UvrAB or RNAP, UvrC, UvrD, DNA Polymerase, Liagse.

UvrAB (NER)

recognizes pyrimidine dimers in DNA sequence. UvrB stays as a marker form UvrB.

UvrC (NER)

interacts with UvrB and cuts the damaged strand

UvrD (NER)

a helicase that unwinds DNA after UvrC cuts out the pyrimidine dimer (bad strand).

5' end of DNA

phosphate group

3' end of DNA

free OH on C3

TRCF

recruits UvrAB during RNA transcription when DNAP recognizes a pyrimidine dimer. Starts NER

NHEJ and HDR

repair double stranded breaks in DNA

Ku

binds to broken ends in NHEJ. Interior of has a positive charge. Is a homodimer.

DNA-PKcs

phosphorylates Artemis activating it

Artemis

is a nuclease that trims back single stranded DNA at the break in NHEJ

NHEJ (non-homologous end joining)

repairs double stranded break either removes a sequence or adds random bases at the break

HDR

repairs double stranded break but uses a sister chromatid to keep bases the same as original. Because it needs sister chromatids it is most active during and after S phase.

MRN and CtlP

nuclease complex that chews back the ends in HDR

RPA protein

protects single stranded DNA and protects it from binding to itself in HDR and lagging strand of replication

BRCA1

helps in end processing

BRCA2

recruits and stabilises Rad51

Rad51

protects single strand and replaces RPA

CRISPR

Clustered Regularly Interspaced Short Palindromic Repeats

CRISPR locus

region in the bacterial genome where CRISPR components are found

CRISPR array

region in the CRISPR locus where viral DNA spacers are stored

Cas genes

CRISPR associated genes that code for proteins in the CRISPR mechanism

tracerRNA

a non coding RNA that is important

Cas 1 and Cas 2

identify the viral DNA and incorporates the sequences into the CRISPR locus as spacers. Cas 1 and 2 always cut viral DNA next to the PAM sequence.

Short palindromic repeats

same forward as it is backward sections of CRISPR locus in between spacers

Cas 9

Associates with a particular crRNA and tracrRNA. Scans DNA for a PAM sequence and binds and unwinds the DNA once it finds it Once crRNA matches with DNA cas9 cuts it.

crRNA

RNA derived from spacer element that matches target DNA to cut

tracrRNA

stimulates cas9 activity

spacer

RNA that comes from viruses

gRNA

a fuse of tracrRNA and crRNA and is man made

RuvC and HNH

mutating these domains of Cas9 makes Cas9 cut 1 strand (nickase), or not cut at all if you mutate both domains.

Cas9 modifications

By tethering other proteins you can change the function of Cas9 to either alter gene expression or mark gene locations.

Nucleotide Base Pairs

A-T, 2 hydrogen bonds.

G-C, 3 hydrogen bonds.

Recognizing 5' to 3'

The first carbon will always be the carbon attached to the nitrogenous base / Guanine.

Then you count clockwise numbering the carbons

So, 5' end is the one that has the phosphorus group attached to the 5th carbon

Guanine

The non-covalent bonds that link DNA strands together occur closest to this.

Prokaryote in Genome and Genes

Genome: typically, one circular chromosome, extrachromosomal elements are also circular.

Genes: 1 gene = 1 protein, no introns, no nucleus.

Eukaryote in Genome and Genes

Genome: many linear chromosomes, mitochondrial and chloroplast DNA is circular

Gene: one gene= can be several, generally related proteins (isoform proteins)

Histone

Proteins that the DNA is wrapped around

Why is Chromatin Strucuture important?

Chromatin structure (euchromatin vs. heterochromatin) relates to how easy it is for a transcription factor to bind DNA and allow expression of the genes downstream.

Ubiquitinylation

Modifies gene expression

Readers

proteins that bind to specific modifications on the histone. Serve as binding site for other proteins, which may be involved in repair, gene expression, or gene silencing

Writers

enzymes that make histone modifications (example = Histone methyltransferase

Erasers

enzymes that remove these modifications (example = Histone demethylase)

Epigenetics

Gene silencing is the basis for this. DNA expression patterns can be inherited. They can also change based on environmental factors.

DNA methyltransferase (DNMT)

Add methyl groups to Cytosine. Occurs in promoters (regulatory regions of DNA). Blocks gene expression

Deoxynucleoside triphosphate (dNTP)

Added to DNA, provides energy to create phosphodiester bond

DNA polymerase

makes phosphodiester bonds and grows DNA strand

leading strand

Goes towards replication fork, away from ORI

Lagging strand

Goes toward ORI

ORI and Replication Fork Steps

1) Separate strands (Helicase)

2) Add a Primer (Primase/DNA polymerase alpha)

3) Elongate DNA strand (DNA polymerase delta or epsilon)

4) Remove primers (DNA polymerase delta or epsilon)

5) Ligate nicked DNA (Ligase)

1. Helicase binds DNA at ORI and separates strands.

2. Primase make RNA primer, DNAP alpha grows primer

3. Primase not needed for leading strand, PCNA clamp adds DNA polymerase epsilon and DNA fragments made.

4. DNA polymerase grows DNA and makes phosphodiester bonds

5A. On the leading strand, DNAP epsilon adds DNA bases

5B. On the lagging strand, primase and DNAP alpha add RNA primers, DNAP delta adds DNA bases

6. Okazaki fragments form

7. DNAP delta removes RNA primer on lagging strand

8.DNA "nick" is sealed by DNA ligase.

9. Topoisomerase keeps Helicase binded to DNA