Cell bio Exam #2

DNA polymerase

The key enzyme that acts as the fundamental machinery and mechanism of DNA replication

Synthesizes DNA

Needs a primer

Gets its energy from breaking phosphates (CTP)

Always adds nucleotides (synthesizes) in one same direction 5’ —> 3’

Fills in gaps that were cut out by exonuclease 

Phosphodiester bond

The covalent bonds that join nucleotides together to form polymers (polynucleotides) Formed between a 3’ hydroxyl and a 5’ phosphate

Formed through dehydration reactions by DNA polymerase

Leading strand

The strand of DNA with longer strands, making synthesis more efficient

Strand that’s synthesized continuously 

Lagging strand

The strand of DNA that is slower to synthesize (needs okazaki fragments) —> discontinuous synthesis

Synthesis is less efficient 

DNA ligase

Enzyme that stitches DNA segments together during synthesis and seals up the space between fragments (In prokaryotes and eukaryotes) 

Okazaki fragments

Small fragments that make up the lagging strand of DNA

Each fragment has its own RNA primer

Primer

Segment made up of RNA that is used to start DNA synthesis

RNA polymerase

Can make a phosphodiester bond and synthesize without a primer —> it can make its own primer

Exonucleases

Enzymes that remove RNA primers by cutting nucleotides (cuts the last nucleotide in a strand) —> only recognizes the end of a sequence of nucleotides (either the 3’ hydroxyl or 5’ phosphate) 

Cleave with directionality

There are 3’-5’ exonucleases and 5’-3’ exonucleases

Endonucleases

Cuts nucleotides within a sequence

Cleave phosphodiester bonds 

DNA polymerase I

In prokaryotes

One enzyme that does 2 jobs in prokaryotes

Acts as a 5’-3’ exonuclease and DNA polymerase

Fills in gaps in DNA sequences between okazaki fragments

*exam question*

DNA polymerase d (delta)

Fills in gaps in DNA sequences between okazaki fragments

5’-3’ polymerase in eukaryotes

Synthesizes the lagging strand of DNA and fills in the gaps between the strands

DNA polymerase III

A polymerase in prokaryotes that synthesizes 99% of the prokaryotic genome 

Synthesizes both the leading strand and the lagging strand

Primase

An enzyme (an RNA polymerase) that makes RNA primers for the leading and lagging strand in prokaryotes and eukaryotes 

Works with DNA polymerase (A-alpha)

DNA polymerase (E-epsilon)

Synthesizes the leading strand of DNA in eukaryotes 

DNA polymerase (A-alpha)

Works with primase to make primers on the lagging strand

Somewhat slow enzyme, so primase speeds up the process to make primers

Helicase

Enzyme that separates double stranded parental DNA at the replication fork

Breaks hydrogen bonds between bases

ATP dependent enzyme —> needs energy to break bonds

Also called DnaB

Catalyzes the unwinding of DNA 

Single-stranded DNA binding proteins (SSB)

Stabilize the unwound DNA, keeping it single stranded so that it can be copied by DNA polymerase

Keeps complementary base pairs from coming back together after being separated by DNA helicase

Keeps dsDNA from reforming until DNA polymerase can bind to the ssDNA

Sliding-clamp proteins

Holds DNA polymerase in place during synthesis

Helps DNA polymerase work at a faster rate (without this protein, DNA polymerase would still be able to synthesize, just at a much slower rate)

Looks like a donut around the DNA —> glides along the DNA, squeezing the DNA polymerase onto the template and holding it in place 

Also called PCNA’s (proliferating cell nuclear antigens) in eukaryotes

Loads DNA polymerase onto the primer and maintains stable association with the DNA template

Helps e and d polymerase synthesize the leading and lagging strand faster

Clamp-loading protein

Proteins that help load sliding-clamp proteins onto the DNA

Opens up the “donut” allowing it to go around the DNA (requires ATP for this) 

Also called RFC’s in eukaryotes 

Topoisomerase (I and II)

Enzymes in charge of making sure that DNA does not get knotted up, and if it does, unwind the knotted DNA) 

Cuts DNA, allows it to unwind, and then re-ligates it back together

Upstream of the replication fork (not exactly on the replication fork) —> un-coiling needs to occur before helicase separates the strands 

Creates a transient (non-permanent) break in the DNA allowing for the free rotation of the DNA strands; then ligates the DNA strands back together once un-knotted

Origin of replication (ori)

Where DNA replication starts (NOT at the replication fork!!!)

Creates 2 replication forks - each going in either direction

In bacteria, there is only 1 short sequence (can be easily put into plasmid vectors) 

In eukaryotes, there are many of these sequences (the sequences eventually run into each other, conjoining)

Initiator

A protein that recognizes the origin of replication in bacteria and starts the DNA replication process by recruiting helicase

Also called DnaA

Works with helicase at the origin of replication (in bacteria) as the key to starting DNA replication

Origin of replication complex

A complex of proteins that recognizes the origins of replications in eukaryotic DNA (Look at image in notes)

Assembly of this complex of proteins to the DNA template leads to the initiation of replication

Made up of ORC subunits

Telomerase

Enzyme that helps to make telomeres

Extends the DNA strand at the end of replication

Type of reverse transcriptase —> synthesizes DNA and simple sequence repeats (uses its RNA as a template to synthesize DNA)

Carries its own RNA template (its RNA is complementary to telomeric simple sequence repeats; allows for the extension of the template strand at the end of the chromosome)

Maintains telomeres at a normal length (stops chromosomes from getting shorter and shorter after each round of replication)

Allows the DNA to be at least as long as it was before (if not longer) after the primers are removed

Protect chromosomes from degradation (via its loop structure) 

Simple sequence repeats

Make up telomeres

Key to maintaining chromosomal length during DNA replication

Braca genes

Breast cancer genes

Genes that delay the cell cycle and detect DNA damage

Pyrimidines

C,U,T (double ringed nucleotide)

Purines

A,G (single ringed nucleotide)

Active site on DNA

At the 3’ hydroxyl

Deamination

A type of DNA damage that often occurs in cytosine —> when an amine group (H2) gets replaced with an oxygen, leading to the base changing from cytosine to uracil (Don’t want uracil in DNA!!!)

Pyrimidine dimer

Neighboring prymadines that become bound to each other where they shouldn’t be, stopping the correct hydrogen bonds to be made —> stops them from being able to hydrogen bond to their complementary base pairs

(Can happen to thymine or cytosine)

Photolyase

An enzyme found in plants that uses light to repair DNA due to exposure to light (ex. pyrimidine dimers)

(Only found in plants)

Mismatch repair

A “DNA repair” mechanism by which cells can repair mismatched base pairs incorporated during DNA replication

Carried out by 3 proteins in bacteria (MutS, MutH, and MutL) —> the change in the shape of the DNA due to the error as well as methyl groups on the DNA allows proteins to recognize the mutated DNA and fix it by cutting out the mistake —> DNA polymerase and ligase then re-synthesize the DNA that was cut out

Carried out by 2 proteins in humans (MutS and MutL) —> similar process to bacteria, however the strand specificity is determined by single strand breaks rather than methylation

MutH

Enzyme that helps with mismatch repair

Cleaves (cuts) the new strand (the one with the mistake) adjacent to the mismatch

Only cleaves opposite of methyl group

(Only in bacteria, because mismatch repair in humans is not dependent on methylation)

MutS

Enzyme that helps with mismatch repair

Identifies and recognizes a mismatch and recruits MutH

Works with MutS to direct excision (removal of nucleotides) between the gap made in the DNA by MutH and the mismatch —> also recruits other enzymes such as helicase to help

(In humans and bacteria) 

MutL

Enzyme that helps with mismatch repair

Works with MutS to direct excision (removal of nucleotides) between the gap made in the DNA by MutH and the mismatch —> also recruits other enzymes such as helicase to help

(In humans and bacteria)

Base excision repair

A repair mechanism for fixing actually damaged DNA (not just a mismatch, a base is actually damaged)

Uses many enzymes such as DNA glycosylase and AP endonuclease

Once the damaged base is taken out along with the AP site, the DNA is re-synthesized by DNA polymerase and ligase

Occurs in bacteria and humans

AP site

A sugar backbone attached to a phosphate with no nitrogenous base attached to it

AP endonuclease

An enzyme that helps with base-excision repair

Cleaves (cuts) the area of the DNA with an AP site (nucleotide without the base)

Nucleotide-excision repair of thymine dimers

A repair mechanism for fixing pyrimidine dimers caused by UV radiation

Uses many different enzymes to excise a primer with a pyrimidine dimer allowing DNA or RNA polymerase to re-synthesize the damaged area

Exinuclease

An enzyme used for nucleotide-excision repair of thymine dimers, which cuts and takes out 2 sides of an oligonucleotide (primer), allowing DNA or RNA polymerase to re-synthesize the area

Homology directed repair

Immunoglobulin gends

B lymphocytes, a type of a that secrete antibodies 

RAG 1 and RAG 2

Recombinase enzymes that recognize short recombination signal sequences

Grab genomic DNA and recombines it at specific sites in introns

Class Switch Recombination

Rearranged VDJ regions combine with different heavy-chain constant chains

Ca, Cu, Cy, Ce

Different types of mammalian constant chain varients

IgM (early), IgG (late), IgE, IgA

Four different classes for mammalian immunoglobulins

IgM

Immunoglobulin class that activates complement, a first line of defense against invading cells or viruses

IgG

Immunoglobulin class that activates compliment and binds to phagocytic cells and can cross the placenta

IgA

Immunoglobulin class in which antibodies are secreted into nasal mucus and saliva to eliminate bacteria and viruses

Found in breast milk

IgE

Immunoglobulin class in whivh antibodies protect against parasitic infections and are involved in allergies

Somatic hypermutation

Produces multiple mutations within rearranged variable regions of both heavy and light chains

Activation-induced deaminase (AID)

Plays a key role in class switch recombination and somatic hypermutation

Gene amplification

Alters genome structure by increasing the number of copies of a gene

RNA polymerase

The principle enzyme responsible for RNA synthesis

Has 5 subunits

Synthesize 5’ —> 3’

Only needs to read one of two strands

Can start the synthesis process without a primer

Makes phosphodiester bonds between nucleotides

Core polymerase

Parts of the polymerase necessary for transcription

Promoter

Region of DNA upstream of the transcription start site (TSS) where RNA polymerase binds to initiate transcription of a gene

E.coli ___ contain -35 and -10 elements

“Upstream”

Something 5’ on a gene

“Downstream”

Something 3’ on a gene

TATA boxes

Element in promoters

Termination signal

Where polymerase stops transcribing (NOT a stop codon)

RNA Polymerase II

Transcribes all protein-coding genes

Synthesizes miRNA and IncRNA

RNA polymerase III

Synthesizes tRNA

RNA polymerase I

Synthesizes rRNA

Transcribes ¾ parts of the ribosome

Mitochondrial RNA polymerase

Synthesizes mitochondrial genes

TFIIx (transcription factor II x)

5 different transcription factors

TFIID

Transcription factor that binds the TATA box and other elements

TFIIB

Transcription factor that recruits RNA polymerase II

TFIIF

Transcription factor that recruits RNA polymerase II

TFIIE

One of the 5 transcription factors (don’t need to know function)

TFIIH

Transcription factor that recruits helicase and CTD kinase

Mediator

Enzyme complex that facilitates interactions between RNA polymerase II and regulatory transcription factors 

Left behind once transcription actually starts

Ribose methylation

Replacing a hydrogen with a methyl group to make rRNA more stable

Base modifications

Stabilizes RNA and reduces immune activation

snoRNPs (Small nucleolar ribonucleoproteins) 

Catalyze ribose methylation and uridine isomerization on pre-RNAs 

Molecule that is part RNA and part protein

tRNA

Can be modified in many different ways

7-methylguanosine cap

A nucleotide that is placed on backward, stopping transcription

Guanylyl transferase

Transfers 7-methylguanosine to where it is needed

Poly-A polymerase

Places poly-A tails where they need to be

Poly-A tail

Regulates translational rates —> gets RNA ready for translation

Regulates mRNA stability

Added to the 3’ end of the RNA

snRNP (small nuclear ribonucleoprotein particles) 

Splice site

5’ or 3’

Where an mRNA strand is spliced

Branch point

Where mRNA is folded up

Spliceosomes 

enzyme that carries out splicing 

made up of RNA’s and proteins —> made up of snRNPs

Processing factor —> when not being used, it hangs out on CTD

Splicing factors

proteins that facilitate splicing by snRNPs by guiding them to the correct splice site

Lac operon

a set of 3 genes expressed as a unit that encodes enzymes that carry out lactose metabolism

In bacteria

Regulates lactose metabolism

z y and a gene

y gene

Lactose permease

Helps lactose get into bacteria by permeating the bacteria’s membrane

Represor protein

Regulates/represses transcription

Only binds to the RNA when lactose is present

Cis-acting element

DNA sequences in the vicinity of the gene that regulate its expression

Trans-acting factors

Factors (ex. proteins) that regulate a genes expression and are encoded by a different gene elsewhere in the genome

CAP protein

Recruits RNA polymerase to the lac operator promoter

Reporter plasmid

Light emitting gene

Mediator complex

Regulates interactions with RNA polymerase II

Upstream regulatory sequences

Bind additional transcription factors that regulate transcription

Must be close to the transcription start sight

Enhancers

Bind transcription factors that regulate transcription, but function independent of proximity and orientation to the transcription start sight

Allows for more “enhanced” transcription

Could be upstream or downstream or backwards and it still works the same because of the way that DNA is a 3D molecule

Many are associated with disease because if there are mutations in these sequences it can cause antibodies to not be made or not work correctly

Cohesion protein

Holds DNA in a specific conformation which allows different proteins such as the enhancer to bind to where it needs to be

Topologically associated domains (TADs)

a DNA model that includes the gene, the promoter region and the enhancer

CTCF

a protein which divides chromosomes into independent domains and prevents enhancers from acting on promoters located in an adjacent domain.

Electrophoretic-mobility shift assay (EMSA)

a technique used to detect protein-DNA interactions