When does DNA replication happen?
In interphase, the portion given as S (synthesis)
What is replicated in DNA replication?
single stranded DNA
What type of replication happens during S-phase and who discovered this?
highly conservative replication/ Meselson and Stahl
How does replication licensing ensure that DNA is replicated only once at each origin per cell cycle?
First origins are licensed meaning they are approved for replication. Then replication machinery initiates replication at each licensed origin, but only functions at the licensed origins.
Leading strand:
the template strand of the DNA double helix so that the replication fork moves along it in the 3' to 5' direction.
Lagging strand:
The lagging strand is the strand of the template DNA double helix that is oriented so that the replication fork moves along it in a 5' to 3' manner.
What are Okazaki fragments and why are they formed?
Okazaki fragments are created during DNA replication because DNA Polymerase can only add nucleotides in a 5' to 3' direction.
Why are RNA primers required for DNA replication?
Initiation of DNA synthesis requires an RNA primer
DNA helicase:
opens DNA strands making them ssDNA
DNA gyrase:
member of the family of topoisomerases; reduce super-coiling of DNA
SSB Proteins:
binds to single-stranded regions of DNA to prevent premature annealing, to protect the single-stranded DNA from being digested by nucleases, and to remove secondary structure from the DNA to allow other enzymes to function effectively upon it.
RNA Primase:
lays down an RNA primer
DNA polymerase I:
removes the primer and replaces it with DNA
DNA polymerase III:
begins replication, laying down nucleotides in a 5'-3' direction.
DNA Ligase:
joins together the newly synthesized strands of DNA by sealing the phosphodiester bonds
What is a telomere and why do chromosomes had them?
A telomere is a region of repetitive nucleotide sequences at each end of a chromatid, which protects the end of the chromosome from deterioration or from fusion with neighboring chromosomes
Where is telomerase expressed?
not expressed in most somatic cells, it is expressed in reproductive cells
What would be the effect on DNA replication of mutations that destroyed DNA polymerase I 3'->5' exonuclease activity?
It will correct mistakes by "backspacing", so if you lose this ability, it will increase the mutation rate because it won't be able to proofread
What would be the effect on DNA replication of mutations that destroyed DNA polymerase I 5'->3' exonuclease activity?
primer would not be removed, replication wouldn't proceed with polymerases in prokaryotes
What would be the effect on DNA replication of mutations that destroyed DNA polymerase I 5'->3' polymerase activity?
eplication wouldn't proceed in eukaryotes because there is no polymerase coming in.
What would be the effect on DNA replication of mutations that destroyed DNA polymerase III 5'->3' polymerase activity?
repair of DNA would be stopped; disrupt the replication process of bacterial DNA not synthesizing new strand
What would be the effect on DNA replication of mutations that destroyed DNA polymerase III 3'->5' exonuclease activity?
errors in bacterial DNA would not be caught and corrected which increases mutation rate/ error rate
Components of typical eukaryotic gene?
TATA box, intron/exon, promoter, poly-tail site, terminator
TATA box:
The TATA box assists in directing RNA polymerase II to the initiation site downstream on DNA.
Exons/Introns:
translated regions/ untranslated regions
Promoter:
help hold the polymerase on DNA sequence that the transcription apparatus recognizes and binds. It indicates which of the 2 DNA strands is to be read as the template and the direction of transcription.
What parts make up the transcriptional unit of the gene?
1. Promotor.
2. RNA-coding sequence.
3. Terminator.
How does DNA give rise to a chromosome from 2nm to 11nm?
double stranded DNA is wrapped about 2 times around a histone octomer and is all held together by an H1 Histone protein
How does DNA give rise to a chromosome from 11nm to 30nm?
nucleosomes wind in a helical fashion to form a solenoid
How does DNA give rise to a chromosome from 30nm to 700nm?
solenoids form a series of loops that are compressed into fibers by the chromosomal scaffold proteins, which are coiled into chromatids
Heterochromatin vs. Euchromatin?
-Heterochromatic is tightly coiled throughout the cell cycle, few active genes, and determines chromosome structure (mainly at centromeres and telomeres) with dark staining regions.
-Euchromatic is less densely packed (tightly coiled only at Metaphase) an
Transcription Basics:
1. Takes place in the nucleus
2. only certain parts are transcribed from ssDNA
3. DNA strand contains a regualtory promoter and a TATA box
4 basic stages of transcription?
1. Recognition
2. Initiation
3. Elongation
4. Termination
Recognition:
recognition of the core promoters/TATA Box
Initiation:
Formation of the preinitiation complex, Binding of RNA Pol II
Elongation:
movement of RNA Pol II to transcribe the molecule
Termination:
STOP, DNA is released (signaled by GC rich regions and Poly-A region)
Where does RNA processing occur?
Inside the nucleus
Steps of mRNA processing?
1.addition of 5' cap
2.addition of 3'poly-A tail
3.splicing
What happens during the addition of the 5'cap?
-Addition of guanosine with 5' to 3' bond
-Methylation
What happens during the addition of the poly A tail?
-Recognition of AAUAA and G/U rich sequence
-Cleavage 20-30 bp downstream of AAUAA
-Polyadenylation
What happens during splicing in mRNA processing?
-RXN 1: the 5' splice site is attacked by the 2'-OH of a conserved unpaired adenosine, resulting in the formation of a 2',5'-phosphodiester linkage
-RXN 2: the free 3'-OH group of the 5' exon attacks the 3'splice site, liberating the circular intron laria
What are the 5 mRNA processing proteins?
1. Capping enzyme
2. RNA methyltransferase
3. Polyadenylate polymerase
4. Poly-A binding protein
5. the spliceosome
Translation basics:
-DNA->mRNA->protein
-Proteins are made of AA connected by polypeptide bonds
-Each protein will have an N terminus and a C terminus
-Synthesis starts with the AA at the N terminus
The genetic code facts:
-written in a linear form (stored in the mRNA)
-coded as triplets (codons)
-unambiguous (1 codon=1 AA)
-degenerate (1 AA can be coded for by more than 1 codon)
-specific start and stop codons (start=AUG, stop=UAA, UGA, UAG)
What is a wobble?
Flexibility in base pairing at the third position of the codon
4 steps of translation and what happens at each?
(1) Activation-5' N terminus amino acid is joined by its carboxyl group to the 3' OH of the tRNA by an ester bond
(2) Initiation-ribosome binds to 5' end of mRNA with the help of initiation factors
(3) Elongation-additional amino acids are added to the po
How does translation differ in prokaryotes vs. eukaryotes?
-initiation codon (AUG) codes for methionine in eukaryotes and for n-formyl methionine in prokaryotes
-transcription takes place in nucleus for eukaryotes and in the cytoplasm for prokaryotes
What is the order of when translation proteins act during translation?
fMET->30S(small subunit)->IF3->70S(Large subunit)->EF Tu->peptidyl transferase->EF G-->RF1
Protein basics:
-an AA consists of an amino group, carboxyl group and an R group
-the R group/side chain distinguishes one AA from another and different R groups have different properties
-Proteins can go through post translation modifications
4 types of AA's:
1. Non-polar (hydrophobic)
2. Polar (hydrophilic)
3. Positive charge
4. Negative charge
4 types of protein structure:
1. Primary-sequence of AA's in the peptide
2. Secondary-configuration in space/interaction between AA's form beta sheets and alpha helices
3. Tertiary-complete 3-D structure that is specific to a given protein
Polar hydrophilic vs. Non-polar hydrophobic groups:
Polar are on the surface and non-polar are on the inside
Mutation Basics:
-Altering protein structure may alter gene function
-different organisms can have different mutation rates
-different regions of the genome can vary in their mutation rates
-Mutations in somatic cells may impact cell phenotypes and can contribute to disea
Base substitution mutation:
involving replacement or substitution of a single nucleotide base with another in DNA or RNA molecule.
Insertion mutation:
the addition of one or more nucleotide base pairs into a DNA sequence.
Deletion mutation:
a part of a chromosome or a sequence of DNA is missing.
Frameshift mutation:
a genetic mutation caused by insertions or deletions of a number of nucleotides in a DNA sequence that is not evenly divisible by three.
Repeat expansions mutation:
responsible for causing any type of disorder categorized as a trinucleotide repeat disorder.
Missense Mutation:
a point mutation in which a single nucleotide is changed, resulting in a codon that codes for a different amino acid.
Nonsense Mutation:
a point mutation in a sequence of DNA that results in a premature stop codon, or a nonsense codon in the transcribed mRNA, and in a truncated, incomplete, and usually nonfunctional protein product.
Transition vs. Transversion:
-Transitions are interchanges of two-ring purines (A G) or of one-ring pyrimidines (C T): they therefore involve bases of similar shape.
-Transversions are interchanges of purine for pyrimidine bases, which therefore involve exchange of one-ring and two-r
What is a loss-of-function mutation?
the result of gene product having less or no function. When the allele has a complete loss of function (null allele) it is often called an amorphic mutation.
What is a gain-of-functino mutation?
changes the gene product such that it gains a new and abnormal function. These mutations usually have dominant phenotypes. Often called a neomorphic mutation.
What is a neutral mutation?
has no harmful or beneficial effect on the organism. Such mutations occur at a steady rate, forming the basis for the molecular clock.
Transposons vs. retrotransposons
-Transposons are DNA sequences that move from one location on the genome to another.
-Retrotransposons are genetic elements that can amplify themselves in a genome and are ubiquitous components of the DNA of many eukaryotic organisms.
What are the flanking direct repeats of transposons?
Flanking repeats are sequences that are repeated on both ends of a sequence, for example, the Long terminal repeats (LTRs) on retroviruses.
What kinds of mutations are likely to arise due to transposition?
spontaneous and induced mutations
Replicative transposition:
old copy stays in place and new copy is made. Always the case for RNA transposons.
Non replicative transposition:
a Transposon copy is excised and moved to another location (jumping genes).
Excision Repair:
works by removing the area of damage. Special enzymes recognize damaged DNA. This repair system comes in two forms: Base-excision repair and short-patch nucleotide excision.
Repair of Mismatched Bases:
is able to identify mismatch errors because such damage leads to a small distortion in the DNA backbone.