Molecular Genetics Exam 2

Define mutation

A stable change in the DNA sequence that can be propagated through replication and cell division

What creates to DNA mutations? (3)

Replication errors, DNA damage, or chromosomal rearrangements if they escape repair

Describe spontaneous mutation

Happens during normal processes at a low rate, higher in eukaryotes than prokaryotes

In base substitution, what is a transition?

When a pyrimidine replaces a pyrimidine or a purine replaces a purine

In base substitution, what is a transversion?

When a pyrimidine and purine are interchanged

What is a frameshift mutation, and why are they so severe?

A change that shifts what three-letter segments are read together. It can uncover a early stop codon, resulting in a truncated protein

What is a trinucleotide repeat (triplet expansion) and what causes it?

3 nucleotide expansion in a repeated sequence, caused by template slippage

Explain Fragile X syndrome

An inherited intellectual disability from too many CGG repeats when they expand during meiosis in females

What are two diseases caused by triplet expansion

Huntington’s disease and Fragile X syndrome

What is the single chromosome mutation when nucleotides are removed?

Deletion

What is the single chromosome mutation when nucleotides are repeated?

Duplication

What is the single chromosome mutation when nucleotides are flipped such that they now read backwards?

Inversion

What is the multiple chromosome mutation when 2 chromosomes exchange nucleotides?

Translocation

What does UV light do that makes it a mutagen?

Causes 2 nearby pyrimidines to link to a pyrimidine dimer

What does ionizing radiation do that makes it a mutagen?

Ionizes water molecules in the cell producing DNA-attacking free radicals that cause double stranded breaks

What happens in the polymerase when the wrong base (dNTP) pairs?

The shape is wrong, so the polymerase can’t close. This slows the rate of catalysis to give time for the wrong base to dissociate

What causes oxidative damage to cells?

Reactive oxygen species are results of normal cellular metabolism and modify nucleotide bases

What is the most common product of oxidative damage?

8-Oxoguanine which results in a G that can bond with C or A bases

What is depurination?

The spontaneous loss of G or A from DNA

What is hydrolytic DNA damage? AKA deamination

Water converting Cytosine to Uracil

What causes deamination and depurination?

They happen spontaneously and commonly without mutagens

What is the purpose of Dam methylase

Recognized GATC site and methylates it. It is also slow and works as a timing mechanism

Why is the hemimethylation of GATC sites by Dam methylase useful in mismatch repair?

The methyl group flags which strand is the original so the incorrect nucleotide is on the non-flagged strand

What does MutS do?

Recognizes alteration in DNA backbone by the bulge created by a mismatch and recruits MutL

How do MutS and MutL do mismatch repair in E. Coli?

MutS binds the mismatch and forms a complex with MutL. They scan bidirectionally forming a loop until they find a GATC site with a methyl tag. They recruit MutH to cleave the unmethylated strand then Helicase II and Pol I exonuclease unwind the DNA from the nick to the mismatch so Pol III can repair the sequence and Ligase seals.

How does mismatch repair in Eukaryotes differ from E. Coli, aside from the use of homologs in place of MutS and MutL?

There is no hemimethylation. MSH recognizes the mismatch then the PCNA clamp identifies which is the nascent strand and exonucleases cleave from the already existing nicks from Okazaki fragments to the mismatch

Why is Pol III used in mismatch repair in E. Coli?

Because the GATC site may be far from the mismatch

What does DNA Photolyase do?

Uses visible light to directly and inexpensively reverse UV-induced pyrimidine dimers in non-mammals

What does Methyltransferase do?

Repairs methylated guanine by taking the methyl group. Suicide enzyme that can only be used once because taking the methyl irreversibly modifies it

What is Base Excision Repair (BER)? (2 steps)

Specific step: base-specific glycosylases scan DNA to remove individual damaged bases such as uracil or 8-oxo-G and leave abasic (AP) sites. General step: AP endonuclease recognizes abasic sites and cleaves the backbone so a Polymerase, Pol I in bacteria, can resynthesize a short segment

What does Nucleotide Excision Repair (NER) do?

Removes bulky lesions

How does Nucleotide Excision Repair (NER) work in E. Coli?

UvrAB scans for distortion then recruits UvrC excinuclease that nicks on both sides of the damage. UvrD removes the segment then Pol I and Ligase repair

What is Xeroderma Pigmentosa and why is it signifigant?

A disease caused by defective NER proteins that means they can’t repair UV damage. Humans don’t have Photolyase and use NER as a major repair pathway. It’s the reason NER proteins in eukaryotes have “XP” names

What is Translesion “bypass” Synthesis?

When Pol III stalls, a translesion Pol can replace it. They have no proofreading and permissive active sites, so they are error prone but keeps DNA synthesis going

What are the 2 types of double-strand repair?

Homologous recombination and non-homologous end joining

What are the fundamental steps of DNA double-stranded break repair? (3)

Processing to create single stranded overhangs. Find homologous DNA to invade with recombinase and form a D-loop. Use the intact DNA as a template to restore missing information

What does the RecBCD complex do?

Binds broken dsDNA ends and unwinds until RecC recognizes a chi site then forms a 3’ overhang

Describe RecA and its function in the dsDNA repair process.

Recruited first by RecB then cooperatively binds more RecA, they coat ssDNA 3’ overhangs, displacing single-stranded binding proteins, forming RecA filaments that promote homology search and strand invasion. Requires ATP to function

There are two ways to complete dsDNA repair, SDSA and DSBR. Describe SDSA (Synthesis-dependent strand annealing).

Helicase melts complementary strand bonds so the new and template strand disassociate, then DNA Pol and Ligase seal the new strands with no recombination

There are two ways to complete dsDNA repair, SDSA and DSBR. Describe DSBR (Double-strand break repair).

The formation of Holliday junctions that must be cut apart resulting in a non-crossover product with a small exchanged segment or a crossover product with mixed strands

What is the RuvAB complex in E. Coli and what does it do? What about RuvC?

It’s a Repairer of UV damage. RuvA recognizes Holliday junctions and RuvB uses ATP to drive branch migration. RuvC cleaves Holliday junctions

When is the recombination resulting from dsDNA repair Holliday junctions an intentional part of the cells lifecycle?

During Meiosis I to promote genetic diversity

What is the name for the type of enzymes that cut Holliday junctions?

Resolvases

What is the key difference between crossover during regular dsDNA repair and crossover during Meiosis?

Repair uses identical sister chromatids while Meiosis uses nonsister chromatids resulting in gene conversion and heteroduplex DNA

What is somatic recombination?

When Mitosis involves nonsister chromatids and results in crossover

When the replication fork arrives before DNA is repaired, what are the 4 possible outcomes?

Translesion synthesis, bypass leaving a gap, gap repair using Holliday junction, or fork regression to rezipper damaged DNA

What happens when DNA Pol conducting replication encounters a nick or gap from ongoing MMR, NER, or BER repair?

The fork collapses and must recover using a variation of dsDNA break Holliday junction repair that recreates the replication fork

What is the problem with nonhomologous end joining?

There is no template so it is usually mutagenic

What is the first problem that must be solved in order to insert human DNA into a bacteria so it will create a desired protein? How is it solved?

Humans have introns that bacteria can’t read. Use transcription to get mRNA where the introns are spliced out, then use reverse transcriptase to convert that RNA into complementary DNA

The poly-A tail creates a complementary poly-T tail that acts as a primer for DNA reverse transcriptase. After replication, reverse transcriptase adds a non-templated poly-C tail to the end of the new DNA strand. What is the purpose of the poly-C tail?

To bind the template switching oligo so the new ssDNA can act as a template for its complement and form dsDNA

What are all of the “ingredients” required for PCR?

DNA template, primers complementary to ends of target, dNTP, DNA Pol, and Mg++

Describe the process of PCR involving multiple temperature adjustments.

High temp to denature DNA, lowered to annealing temp so primers attach, keep at room temp for elongation, then repeat

What is the purpose of giving limited replication time during PCR?

So each round increases “focus” on the region of interest. Every round after round 3 doubles the molecule of interest

What is Gel electrophoresis?

Separates DNA fragments by length, or shape for circular DNA. Uses EtBr intercalator to visualize DNA progress across the gel under UV

What primers do you need for PCR?

A foward primer and a reverse primer that is the reverse complement of the forward primer

What are the 5 steps of cloning?

Obtain DNA segment. Select a vector, typically a plasmid, capable of self replication. Join DNA fragments covalently to insert desired sequence. Transform recombinant DNA into a host. Select for hosts that contain the recombinant DNA.

In a cloning vector, what is a polylinker?

The insertion site for foreign DNA

In a cloning vector, what is the selectable marker?

Something to distinguish successful recombinant DNA vectors, usually antibiotic resistance

In a cloning vector, what is the significance of the origin of replication?

Determines a high or low copy number

In cloning, what is a shuttle vector?

The plasmid backbone a desired gene is placed in to inject it into bacteria

In a cloning vector, what is a repressor?

Inducible genes that control the level of expression

Describe the process of Sanger sequencing.

Using ddNTPs which lack a 3’ OH, DNA fragments of varying length are formed. These are denatured and then can be run through polyacrylamide gels or use a dye and laser to convert to a color sequence

What is the benefit of the modern system where nucleotide-specific dyes and a laser are used to sequence DNA over the polyacrylamide gels?

It can more easily detect heterozygotes

What is Real-time PCR (qPCR)?

Uses florescence to measure PCR as it happens. Tracks how early of a cycle you cross the threshold into exponential growth. The earlier, the more template DNA is present

After your protein clone is produced, what do you do?

Use SDS gel electrophoresis to denature proteins and separate them by size. Use Western blotting or Antigen-based methods to attach a signal antibody to desired proteins. Then attach a GST handle to purify the protein out of the solution

What are florescent tags used for and how are they used?

To detect where something is in a cell and whether it is active. It’s DNA code can be inserted after the desired protein so that they are transcribed and produced together as tagged proteins

What is 1 centimorgan (cM) in recombinant mapping?

Represents a 1% chance of recombination during meiosis, used to map relative distances of alleles on a chromosome

What is an SDS site and how is it used in reassembling a genomic sequence?

A sequence-tagged site is an area that can be easily amplified during PCR. Overlaps between SDS sequences from different fragments are used to order the DNA map

What is cytogenetics?

The microscopic study of chromosomes

In cytogenetics what is FISH?

Florescent in-situ hybrid, cloned DNA with dye that hybridizes with other chromosomes to reveal chromosome locations

What is the order of fragment assembly in DNA sequencing?

Overlapping fragments form Contigs, then Scaffolds, then Chromosomes

What is the relationship between Genetic maps and Physical maps of a genome?

The order of markers is the same but physical distance doesn’t equal the percent recombination map distance because recombination rates are influenced by other factors like chromatin structure

When would whole genome shotgun sequencing be used over ordered clone sequencing?

For small genomes with few repetitive sequences

What are genome-wide association studies?

Comparing the DNA of a diseased population to a healthy population to identify any SNPs that could be related to the disease

Define synteny

Organisms with recent evolutionary divergence show regions of similar gene content and organization

Define evolution.

The change in inherited characteristics of populations over generations

What percentage of DNA do humans share with chimps, neanderthals, and other humans respectively?

99.0% with chimps, 99.7% with neanderthals, and 99.9% with other humans

Describe the amount and effect of neanderthal DNA in modern humans.

Most humans have 1-3% neanderthal DNA representing a cumulative 30% of the neanderthal genome. This results in diseases like Type II diabetes

What is the genetic contribution of the extinct hominid Denisovans to the modern human genome?

Their genome makes up 5% of aboriginal DNA including the high altitude adaptation in Tibetans

Define haplotype.

A set of DNA variants that tend to be inherited together due to proximity on the chromosome, used to map migrations of humans out of Africa

Describe the “Next Generation” sequencing tecnique.

Uses terminator nucleotides to only allow one nucleotide addition at a time and dyes to color-code nucleotides, then immobilizes DNA fragments on a surface to take pictures as replication occurs. The analysis of the color changes results in a sequence. It is fast and large scale but struggles with repetitive sequences

Describe the “Third Generation” sequencing technique.

Nanopore sequencing where helicase is fuse to a pore that DNA is unwound into. The pore reads electromagnetic currents to identify the passing nucleotide, allowing reading without breaking DNA into segments and so is good for repetitive sequences

6 key facts about RNA transcription

No primer, requires Mg2+, synthesizes RNA from 5’-3’, copies template DNA 3’-5’, polymerizes rNTPs, and starts upstream of a start codon creating 5’ UTR sites

What is the function of 5’ UTR sites in RNA transcription

They help initiate translation, influence translation efficiency, and impact RNA stability

What are the main functions of each of the RNA polymerases found in eukaryotes?

Pol I makes rRNA, Pol II makes mRNA, and Pol III makes tRNA

The overall structure of RNA Pol is conserved between bacteria and eukaryotes. What is the structure?

5 subunits and a sigma factor, the active site has Mg2+ ions

What does Actinomycin D do and why might you want to use it?

Intercolates in DNA inhibiting RNA and DNA synthesis by preventing binding, used in chemotherapy

What does α-amanitin do and where is it found in nature?

Selectively inhibits RNA Pol II by directly binding RNA, found in deadly mushrooms

What is the purpose of the sigma factor in RNA synthesis?

It determines where to start and which direction to go

What are the RNA promoter sites in bacteria?

A -10 site in all for specificity and initiation, a -35 site in most to make the bond stronger, some have a -40 to -60 region called the “UP-element” also for added strength

Why are there multiple rounds of abortive initiation in RNA transcription before the RNA gets over 10bp long?

A growing mRNA chain is unstable