Molecular Cell Biology - Spring 2022

How large is our genome? How many base pairs does it contain? How long is it when fully extended? (part1)

Our genome is 3200 millions of base pairs big. It contains 3 × 109 base pairs. It is about 20,000 phone books long

What is Chromatin?

Chromatin is the fibrous complex of eukaryotic DNA and histone proteins.

-What is Histones?

Histones are the protein components of chromatin. The main histones are H1, H2A, H2B, H3, and H4.

-What's the charge (polarity) of Histones? What amino acid residues are responsible for that net charge? Why is that important?

The charge of histones is positive. The amino acid residues that are responsible for that net charge are lysine and arginine. It's important because lysine and arginine are positively-charged amino acids that can bind to the negatively-charged DNA molecule.

What does Micrococcal Nuclease do to chromatin?

Micrococcal nuclease partially digests the chromatin to obtain gel electrophoresis of DNA fragments.

What type of nuclease is Micrococcal Nuclease? Is it specific for DNA or can it cut RNA as well?

Micrococcal nuclease digests chromatin. Micrococcal nuclease is an enzyme that degrades DNA. It yields ~200bp fragments.

-By the way, what are exonucleases? And how about endonuclease? What's the difference? If you have a "nick" in the DNA (a break in one of the strands of the DNA), and you want to "chew up" the DNA starting at that point, what kind of nuclease activity would be needed, an endonuclease or an exonuclease? Why?

Exonuclease is an enzyme which removes successive nucleotides from the end of a polynucleotide molecule. Endonuclease is an enzyme which cleaves a polynucleotide chain by separating nucleotides other than the two end ones.

Exonucleases "chew" the ends of a DNA strand. Endonucleases "chew" within the strand of DNA. An endonuclease would be needed because since one of the DNA strands broke within itself, endonucleases can "chew" within the DNA.

What's a Nucleosome? How many base pairs (bp) of DNA are contained in a Nucleosome?

A nucleosome is the basic structural unit of chromatin consisting of DNA wrapped around a histone core. It has 200 base pairs.

What's a linker protein?

A linker protein is a nonhistone protein that attaches to the linker DNA in between nucleosome core particles.

What's a Nucleosome Core Particle? How long is it (in bp)?

A nucleosome core particle is a particle containing 146 base pairs of DNA wrapped around an octamer consisting of two molecules each of histones H2A, H2B, H3, and H4.

What's a Chromatosome? How long is it (in bp)?

A chromatosome is a chromatin subunit consisting of 166 base pairs of DNA wrapped around a histone core and held in place by linker histone.

What happens to chromatin if you allow its digestion with Micrococcal Nuclease for extended amounts of time?

What would happen if you allow the digestion to go on for several hours? How may bands would you get? -How many copies of each histone are there in each of the above structures?

In each chromatosome, two each—H2A, H2B, H3, and H4, one H1

Different levels of chromatin structure:

1 st order - "Beads on a String" or 10 nm fiber

2 nd order - Chromatin fiber or 30 nm fiber - can be in the solenoid or zigzag form

3 rd order - Looped form

4 th order - Minibands (in Heterochromatin and in condensed chromosomes during Metaphase)

-Is this hierarchical model considered correct nowadays? If not, what's the current model for large scale chromatin organization? -What are TADs? -What protein(s) play a role in each of the above structures?

Core histones H2A, H2B, H3, H4 in first order; Core histones and H1 in second order; core histones, h1, and scaffolding protein loop in third order; core histones, h1, and scaffolding protein loop in third order and minibands in foruth order

What's heterochromatin? What's euchromatin?

Heterochromatin is condensed, transcriptionally inactive chromatin. Euchromatin is decondensed, transcriptionally active interphase chromatin.

-What's constitutive heterochromatin? What's facultative heterochromatin?

Constitutive heterochromatin is always condensed. Facultative heterochromatin is not always condensed because it depends on the cell and phase of the cell cycle it is in.

-During what stage of the cell cycle is chromatin fully packed? Is there any transcription at that time? Why?

Metaphase is the stage of the cell cycle that chromatin is fully packed. There is no transcription at that time because the DNA is not accessible and inactive.

What's a Centromere? What's the Kinetochore?

A centromere is a specialized chromosomal region that connects sister chromatids and attaches them to the mitotic spindle. A kinetochore is a specialized structure consisting of proteins attached to a centromere that mediates the attachment and movement of chromosomes along the mitotic spindle.

What's a Telomere?

A telomere is a repeat of simple-sequence DNA that maintains the ends of linear chromosomes.

What's the main role of Centromeres?

The main role is ensuring the correct distribution of duplicated chromosomes to daughter cells during mitosis.

Why are Telomeres important? What type of sequence is found at the Telomere? Why is that relevant?

Telomeres are important because they play critical roles in chromosome replication and maintenance. Telomeres consist of repeats of a simple-sequence DNA containing clusters of G residues on one strand. It's relevant because it forms loops at the ends of chromosomes and bind a protein complex that protects the chromosome termini from degradation.

What's the only shared feature of all eukaryotic Centromeres?

The only shared feature is CENP-A.

Are there any genes located in Telomeres? How about Centromeres? Why?

There are no genes located in telomeres and centromeres because they do not have genetic function and do not encode for any gene product.

-What characterizes centromeric regions in humans? Is there any feature that allows their identification?

Specific DNA sequences have not been identified that can identify centromeres. CENP-A alters chromatin structure which appears to be the primary determinant for centromere function and identity

The complexity of eukaryotic genomes. PART 2 of the study guide.

Is there a direct correlation between genome size and biological complexity?

No, there is no direct correlation between genome size and biological complexity.

Is there a direct correlation between genome size and number of genes?

No, there is no direct correlation between genome size and number of genes.

How many genes are there in our genome?

There are about 25,000 genes in our genome.

What's a gene?

A gene is a segment of DNA expressed to yield a functional product.

In genomic terms, how closely related are we with other members of our own species?

We are 0.1 % closely related with other members of our own species.

What is our genome made of?

Our genome is made of introns, repetitive DNA sequences, duplicated DNA sequences, pseudogenes, non-repetitive spacer sequences, 5' UTR and 3' UTR (untranslated regions), and protein coding sequences.

What's an intron? How long can they be? How many introns are there per gene? What's their average size? What's an exon?

An intron is a noncoding sequence that interrupts exons in a gene. They can be about 52,000 base pairs long. A gene can contain 27,000 base pairs of introns. Their average size is about 1000-3000 base pairs. An exon is a segment of a gene that is included in a spliced mRNA.

What's alternative splicing? Why is it important?

Alternative splicing is the generation of different mRNAs by varying the pattern of pre-mRNA splicing. It's important because the presence of introns allows the exons of a gene to be joined in different combinations, resulting in the synthesis of different proteins from the same gene.

What's the most frequent type of sequence component of our genome?

The most frequent type is repetitive DNA sequences.

How many different types of repetitive elements exist?

There are 3 different types: simple-sequence repeats, retrotransposons, and DNA transposons.

What's the difference between a retrotransposon and a DNA transposon? Which is more abundant in our genome? Why?

Retrotransposons are mediated by reverse transcription whereas DNA transposons move the genome by being copied and reinserted as DNA sequences, rather than moving by reverse transcription.

What are SINEs? What are LINEs? What's the main difference between them?

SINEs (short interspersed elements) and LINEs (long interspersed elements) are members of a family of highly repeated retrotransposons in mammalian genomes. The main difference between the two are that SINEs do not encode proteins and have a shorter base pair length whereas some LINEs can encode proteins and have a longer base pair length.

Which ones are the most "damaging", SINEs, LINEs, or DNA transposons? Why?

LINEs are the most damaging because it can be able to move different sites in genomic DNA, introducing variability and instability in our genome.

Why are humans so different from other animals? Is that due to the number of genes?

Humans are different from other animals because of what is in human genes, not the number of genes.

Why do we say that DNA replication is semi-conservative?

It's semi-conservative because each parental strand serves as a template for the synthesis of a new complementary daughter

What are the two features shared by all DNA Polymerases?

The two features are that all polymerases synthesize DNA only in 5' to 3' direction and can add a new deoxyribonucleotide only to a preformed primer strand that is hydrogen-bonded to the template.

How does DNA Polymerase know where to begin replication?

DNA polymerase knows where to begin replication by finding the origin of replication.

What's an origin of replication? How many are there in the human genome? Why is that important at all?

An origin of replication is a specific DNA sequence that serves as a binding site for proteins that initiate replication. There are 30,000 in the human genome. It's important because the process of DNA replication can perform faster and efficiently.

What's a helicase? What role do they play during replication?

A helicase is an enzyme that catalyzes the unwinding of DNA.

What's an ORC? What role do they play during replication?

An ORC (origin recognition complex) is a protein complex that initiates DNA replication in eukaryotic origins.

How is the replication of eukaryotic DNA different from that of prokaryotic DNA?

Eukaryotic DNA replication has multiple origins of replication. Prokaryotic DNA replication has only one origin of replication.

What's a replication fork?

A replication fork is the region of DNA synthesis where the parental strands separate and two new daughter strands elongate.

What's the role of RPA? What kind of protein is RPA?

RPA (replication protein A) is a single-stranded DNA-binding protein that stabilizes the unwound template DNA to keep it in an extended single-stranded state so that it can be copied by the DNA polymerase

Why is it important to have Primase? Can you replicate DNA without Primase? In terms of their need for Primase, is there any difference between lagging and leading strand replication?

It's important to have primase because it's one of the two things needed to start replication. Yes, there are differences between lagging and leading strand replication because the leading strand can already be replicated due to its 5' to 3' direction while the lagging strand will need further processing of replication due to the 3' to 5' direction it's going.

What are the two main types of DNA-Polymerases involved in DNA replication? What does each one do?

A / DNA-Pol α - works with Primase starting DNA replication. It has low fidelity and low processivity (What is processivity? What is fidelity?)

DNA-Pol δ / ε are the main replicating DNA Polymerases, being the high processivity/high fidelity enzymes that do the bulk of the work.

Fidelity - the accuracy of DNA replication

Processivity - the number of nucleotides incorporated by a polymerase before it dissociates

How do you load the DNA-Pol δ onto your DNA? What's RFC? What's PCNA? What do they do?

To load the DNA polymerase δ onto DNA, RFC and PCNA are needed. RFC (Replication Factor C) is a clamp loading protein. PCNA (Proliferating Cell Nuclear Antigen) is a sliding clamp protein. They both help clamp the DNA polymerase to initiate the process of DNA replication.

Why are Ligases needed during DNA replication? What do they do?

Ligases are needed because they join DNA fragments together to form a new intact DNA strand.

What's an Okazaki fragment?

An Okazaki fragment is a short DNA fragment synthesized to form the lagging strand of DNA.

How are the RNA fragments produced by Primase eliminated from the DNA? Which enzyme does the job?

The RNA fragments are removed by the enzyme RNase H.

Why are Topoisomerases needed? What do they do? How many different types of topoisomerases are there?

Topoisomerases are needed because they catalyze the reversible breakage and rejoining of DNA strands. There are two types of topoisomerases: Type I (breaks on one strand only) and Type II (breaks on both strands).

How does DNA-Polymerase achieve high fidelity? What is the "proofreading" activity?

DNA polymerase can achieve high fidelity by checking the conformational changes of the DNA polymerase and proofreading by the DNA polymerase. The "proofreading" activity is the selective removal of mismatched bases by DNA polymerase.

What's the normal frequency at which DNA-Pol δ introduces mutations in the DNA during replication?

The normal frequency is every 100 nucleotides of newly synthesized DNA.

Telomeres & DNA Repair part 3.

How are telomeres replicated? What's the enzyme that maintains the length of the telomere? Is it made in all eukaryotic cells?

The telomeres are replicated by using the template RNA to allow telomerase to generate multiple copies of the telomeric repeat sequences, thereby maintaining telomeres in the absence of a conventional DNA template to direct their synthesis. The enzyme that maintains the length of the telomere is Telomere Repeat Binding Factor (TRF). Yes, it is made in all eukaryotic cells.

What is the enzyme that responsible for extending the 3' end of telomeres? How does it achieve this feat?

The enzyme responsible for extending the 3' end of telomeres is RNA primer. It's achieved by removing the RNA primer from the DNA.

Why is telomeric length considered to be a "biological time keeper"? What happens when telomeres get too short?

Telomeric length is considered a "biological time keeper" because telomeres gradually shorten as cells age. When telomeres get too short, the cells go either into SENESCENCE (a stage of diminished metabolic activity characterized by the inability of the "senescent" cell to go through additional rounds of cellular replication), or APOPTOSIS (a type of cell death that is programmed within our genome; also known as "programmed cell death").

What could happen if telomerase expression is disregulated? Are telomerase levels higher or lower than normal in cancer cells? How about in patients with certain types of progeria?

If telomerase expression is disregulated, there can be a high rate of telomerase loss or have higher levels of telomerase due to a mutation the telomerase has. In cancer cells, the telomerase levels are higher than normal. In patients with certain types of progeria, the telomerase levels are lower than normal.

How do DNA mutations occur?

DNA mutations occur by incorporation of incorrect bases during DNA replication or chemical changes.

What's a silent mutation?

A silent mutation is a change that occurred in the nucleotide sequence of genomic DNA that has no effects on the type of genetic information encoded in the sequence

Why are mutations important? Do they play any role in evolution?

Mutations are important because they play a role in the evolution of a species by incorporating genetic variation. To maintain the integrity of a species genome, cells have therefore had to evolve mechanisms to repair damaged DNA.

What's the most common type of mutation? A/Formation of a cyclobutane ring between adjacent Thymidines due to exposure to ultraviolet radiation

The most common type of mutation is exposure to radiation or chemical changes.

What are the two main types of spontaneous mutations? Why are they important?

The two main types are deamination and depurination. They are important because it creates a variation to the genomic DNA.

What's the most efficient mechanism to fix cyclobutane rings in nature?

The most efficient mechanism is nucleotide excision repair.

Is that mechanism present in placental mammals (including humans)?

Yes, the mechanism is present in placental mammals.

What's the first thing that must be done before fixing any given mutation? Why is this so important?

The first thing that must be done is to recognize the strand that has the mutation. It's important because without recognizing and not correcting the mutation, the mutation will remain preserved

What are two examples of direct reversal of DNA damage? Which one is present in humans?

Two examples are direct repair of thymine dimers by photoreaction and direct repair of O6-methylguanine. The direct repair of O6-methylguanine is present in humans.

What is Excision Repair?

Excision repair is a more general means of repairing a wide variety of chemical alterations to DNA.

Identify the two enzymes that are ALWAYS involved in Excision Repair

The two enzymes are DNA polymerase and ligase.

Which ones are the genes mutated in Xeroderma Pigmentosum (XP)?

The genes mutated in XP are XPA, XPB, XPC, XPD, XPE, XPF, and XPG.

During Nucleotide-Excision Repair, identify the role played by the following proteins: TFIIH (contains XPB and XPD, which are helicases), XPC (C: Seeks the damage, it is the main protein involved in damage identification), RPA (what's the other process in which RPA plays an important role?), XPG and XPF (endonucleases), DNA Polymerase, and Ligase.

TFIIH unwinds the DNA; XPC recognizes the disrupted base pairing in a DNA strand; RPA helps bind TFIIH to the DNA strand where the disrupted base pairing is found and is involved in transcription-coupled repair; XPG and XPF cleave the DNA on the 5' and 3' sides of the damaged site, which leaves a gap; DNA polymerase fills the gap; ligase seals the gap

What's the difference between transcription-coupled repair and nucleotide excision repair? At the protein level, which ones are the proteins that are different between one mechanism and the other? Which proteins are shared between the two?

The difference is that transcription-coupled repair is dedicated to repairing damage within actively transcribed genes. Nucleotide excision repair removes damaged bases from a DNA molecule. At the protein level, transcription-coupled repair contains CSA and CSB whereas nucleotide excision repair contains XPC. They both share XPA, XPB, XPD, XPE, XPF, XPG, and RPA

Mismatch repair is another type of excision repair. What human disease is associated to mutations affecting enzymes involved in this repair mechanism? Which enzyme is the one that makes a nick in the damaged DNA in bacteria? Is this enzyme present in humans?

Hereditary NonPolyposis Colorectal Cancer (HNPCC) is the human disease associated in the repair mechanism. The enzyme responsible is MutS. This enzyme is present in humans

What's the mechanism involved in translesion DNA synthesis? What's the outcome of translesion DNA synthesis? Is any other repair mechanism required after translesion DNA synthesis? Why? By the way, can translesion DNA be considered to be a true DNA repair mechanism? Why?

The mechanism is where the cell can bypass DNA damage at the replication fork, which can then be corrected after replication is complete. The outcome is that it can either sometimes it can leave the mutation or repair the mutation. Nucleotide-excision repair is required after translesion DNA synthesis to repair the mutation. Translesion DNA synthesis cannot be considered to be a true DNA repair mechanism because it continues synthesizing DNA without fixing the mistake first like the other repair mechanisms perform.

How do you introduce variability into our DNA? (Part 4)

What are the two main types of homologous recombination?

The two main types are general homologous recombination and site-specific recombination.

What's the most important difference between those two types of recombination?

General homologous recombination requires extensive (long) regions of sequence identity, whereas site-specific recombination requires only short stretches of sequence identity (hence, site-specific recombination is fully dependent on proteins that identify the regions of sequence identity and drive the recombination event).

When does homologous recombination take place? What is it important for in biological terms?

Homologous recombination takes place from the breakage and rejoining of two parental DNA molecules. It is important because it leads to the reassortment of the genetic information of the two parental chromosomes.

Can homologous recombination be considered to be a DNA repair mechanism? Why?

Yes, homologous recombination can be considered to be a DNA repair mechanism because it repairs the double strand break found in the DNA.

What is a Holliday junction?

A Holliday junction is a mobile junction between four strands of DNA.

What are the two possible outcomes of resolving a Holliday junction?

The two possible outcomes are isomerization and resolution.

What's the first event during general homologous recombination? What type of enzyme would be responsible for that first event?

The first event is the exchange of DNA between chromosomes without altering the arrangement of genes within the genome. An endonuclease would be responsible for that first event.

What's the role of RecA? What's the role of Rad51?

RecA is the key protein involved in the central steps of homologous recombination. Rad51 is a eukaryotic protein that functions similarly to RecA in homologous recombination.

What's the role of RuvA, RuvB, and RuvC?

RuvA, RuvB, and RuvC resolve Holliday junctions

Name the most important proteins involved in antigen recognition in the adaptive immune system?

The most important proteins are T cell receptors and immunoglobulins.

How many different protein chains form an immunoglobulin?

Two chains are formed: a heavy chain protein and a light chain protein (each is present in two copies per immunoglobulin molecule, that is, there are 2 copies of the heavy chain and 2 copies of the light chain).

How many different types of antibodies are produced by each B cell?

The B cell produces 4 different types of antibodies (IgM, IgG, IgE, and IgA).

How is the incredible diversity of antibodies generated?

It is generated after the formation of rearranged immunoglobulin genes by two processes that occur in only B lymphocytes: class switch recombination and somatic hypermutation.

How many gene segments are used to code for the light-chain? How many different types are there for each one of those segments?

There are three segments: V, J, and C. There are 250 different V segments, 4 different J segments, and only one C segment, for a total of 1,000 different combinations.

How many different gene segments are used to code for the heavy-chain? How many different types are there for each one of those segments?

There are four segments: V, D, J, and C. There are 500 different V segments, 12 different D segments, 4 different J segments, for a total of 24,000 different combinations. Additionally, there are 5 major types of C segments. Only one C segment is used at a time and different C segments are used during the maturation of the immune response.

What are the enzymes involved in the process of site-specific recombination in Lymphocytes? What is the role of the RAG genes? What's the role of the Terminal Deoxynucleotide Transferase? What's the role of AID?

The enzymes are Ig and T-cell receptors. RAG (Recombination Activating Genes) genes trigger the binding of the V and D segments selected during the process and that also enhance the cleavage and rejoining of the broken coding strands. Terminal Deoxynucleotide Transferase (TDT) adds nucleotides at will. AID (Activation Induced [Cytosine] Deaminase) deaminates cytosine residues in the region where the VDJ gene segments meet the gene segment coding for the constant region, over a short sequence known as the "switch" region (S region)