We Need Nucleic Acids!

Chapter 25 Homework Assignment • The following problems will be due once we finish the chapter:

4 5, 4, 5 8, 8 9, 9 11

DNA Protein Protein Trait DNA RNA mRNA RNA

rRNA

Pol

tRNA

• DNA contains genes, the information needed to synthesize functional proteins and RNAs – DNA also l contains t i segments t th thatt play l a role l iin regulation l ti off gene expression (promoters, operators, etc.) • Messenger RNAs (mRNAs) are transcribed from DNA by RNA polymerases and carry genetic information from a gene to the ribosome complex

Minimal Coverage of Section 25.3

• Ribosomal RNAs (rRNAs) are components of the ribosomes and play a role in protein synthesis in conjunction with the template mRNA and the AA carrying tRNA • Transfer RNAs (tRNAs) carry the AAs designated by the codons of the mRNA and bind to the Ribosome to help form the growing polypepide chain

Chapter 25

1

Chapter 25

3

Chapter 25

Nucleotides and Nucleic Acids

DNA Metabolism

Nucleotides are the building blocks of nucleic acids A nitrogenous base A phosphate group

(pyrimidines or purine)

A pentose sugar DNA Polymerase III

Nucleotides have three characteristic components Chapter 25

4

1

Structure of a Nucleotide

Nucleic Acid Structure

Major Bases in Nucleic Acids

DNA Strands

• The bases are abbreviated by their first letters (A, G, C, T, U). • The purines (A, G) occur in both RNA and DNA

• The opposing strands of DNA are not identical, but are complementary. • This means: they are positioned to align complementary base pairs: – C with G, and A with T, but the strands run antiparallel to each other

• You can thus predict the sequence of one strand given the sequence of its complementary strand.

• The pyrimidine C occurs in both RNA and DNA, but

• Note that sequence conventionally is written from the 5' to 3' end

• T occurs only in DNA, and U occurs only in RNA Chapter 25

• Such a structure is useful for information storage and transfer! 5

Chapter 25

Nucleic Acid Structure

Nucleic Acid Structure Stabilization of Double Helix

DNA

• Weak forces stabilize the double helix:

• DNA consists of two helical chains wound around the same axis in a right-handed fashion aligned in an antiparallel fashion.

(1) Hydrophobic Effects: Burying purine and pyrimidine rings in the interior of the helix excludes them from water (2) Stacking interactions: Stacked base pairs form van der W l contacts Waals t t

• There are 10.5 base pairs, or 36 Å, per turn of the helix.

(3) Hydrogen Bonds: H-bonding between the base pairs (not on the backbone!)

• Alternating deoxyribose and phosphate groups on the backbone form the outside of the helix.

(4) Charge-charge interactions: Electrostated repulsion of negatively charged phosphate groups is decreased by cations (e.g. Mg2+) and cationic proteins

• The planar purine and pyrimidine bases of both strands are stacked inside the helix. Chapter 25

7

6

Chapter 25

8

2

DNA Metabolism

Nucleic Acid Structure

The BIG Picture

Interstrand H-bonding Between DNA Bases

• DNA is the molecular vehicle used for the stable storage of genetic information • Stable does not mean static however! • DNA metabolism encompasses: – Processes that yield faithful copies of DNA molecules (aka. Replication) – Processes that affect the inherent structure of the information (aka. Repair and Recombination)

What do you think is the most important requirement for any copy of DNA?

Watson-Crick base pairing Chapter 25

9

Chapter 25

11

DNA Metabolism

Nucleic Acid Structure

Gene Naming – An Aside

DNA Structure Summary

• Most of what we now know about the replication process was gleaned by observation of bacterial systems (WHY?) • In this chapter, you will see the names of numerous genes and their products • Bacterial genes are usually names using three lower case, italicized letters that often reflect the gene gene’s s apparent function. • If more than one gene affects the same process the letters A, B, C, etc. are added – dnaA: Gene product that affects DNA replication – recB: Gene product that affects recombination

• Bacterial proteins often retain the name of their genes, except you capitalize the first letter and lose the italics Chapter 25

10

Chapter 25

12

3

DNA Metabolism

DNA Replication Replication is Semi-conservative • Similar to what we see in PCR, each strand of the duplex DNA serves as a template for the synthesis of a new DNA strand

Chapter 25

• The use of these “old” DNA strands as templates produces two “new” new DNA strands that are duplexed with their template.

Chapter 26

• This process yields four DNA strands in two “old/new” duplexes Chapter 27

• This is semi-conservative replication What do you think conservative replication would be?

Chapter 25

13

Chapter 25

15

DNA Replication

DNA Replication

Replication Follows a Set of Fundamental Rules

Replication is Semi-conservative

• DNA replication follows a set of fundamental rules that were determined based on early research with bacterial DNA processes: (1) Replication is semi-conservative (2) Replication begins at an origin and usually proceeds bidirectionally

• This hypothesis (proposed by our good friends Watson and Crick) was verified by Meselson and Stahl in their CsCl gradient experiment.

(3) DNA synthesis proceeds in a 5’ → 3’ direction and is semi-discontinuous

Chapter 25

14

Chapter 25

16

4

DNA Replication

DNA Replication

So how does Replication start and/or run?

Now the strands are open, what next?

• Once semi-conservatism was confirmed several other questions came up: – Are the parent DNA strands completely unwound before replication? – Does replication begin at random points or a unique site? – Once initiated, does replication move in uni- or bidirectional directions?

Chapter 25

17

Chapter 25

19

DNA Replication

DNA Replication

Replication begins at an Origin

DNA Synthesis Proceeds 5’ → 3’

• To be used as a template, the double helix must first be opened up and the two strands separated to expose unpaired bases. • The positions at which the DNA helix is first opened are called replication origins • IIn simple i l cells ll lik like th those off b bacteria t i or yeast, t origins are specified by DNA sequences several hundred nucleotide pairs in length. • This DNA contains short sequences that attract initiator proteins, as well as stretches of DNA that are especially easy to open.

What bases do you think are present at the origin? Chapter 25

• The opening of the duplex puts a “bubble” of single stranded DNA with two replication forks ready for strand y synthesis • John Carins demonstrated experimentally that synthesis of the new DNA strands is simultaneous and bidirectional

18

• A new strand of DNA is always synthesized 5’ to 3’ with the new dNTP being added to the 3’ – OH group • The leading strand is the template that is read from its 3’ end to its 5’ end. • The lagging strand is read 5’ to 3’. What does that mean for the synthesized strand? Uh oh. • Reiki Okazaki determined that the lagging strand is in fact synthesized discontinuously in pieces called Okazaki fragments. • These fragments are connected later by another enzyme. Chapter 25

20

5

DNA Replication

DNA Replication

Who does all the work?

DNA Polymerase

• Enzymes of course! • Nucleases:

• The general DNA Polymerase reaction is a nucleophilic attack of the 3’ – OH group (the nucleophile) on the α phosphate group of an incoming deoxynucleoside

– Catalyze the hydrolysis of DNA (DNases) or RNA (RNases)

Hydrolysis of PPi will give us another 19 kJ/mol of energy to push p sh the reaction

– Either externally (exonucleases) or at internal sites (endonucleases)

• Polymerases: – C Catalyze t l th the fformation ti off a phosphodiester h h di t bond b db between t th the 5’ – phosphate and the 3’- OH

(dNMP)n + dNTP → (dNMP)n+1 + PPi – Requires a primer, a short strand of DNA (or RNA) that has a free 3’ – OH group for elongation

• All the other players: – Helicases, Ligases, Topoisomerases, DNA binding proteins, and many more! Chapter 25

21

23

DNA Replication

DNA Replication

DNA Polymerase

DNA Polymerase

• Prokaryotes and Eukaryotes actually possess several distinct DNA Polymerases • They all catalyze the same fundamental reaction, a phosphoryl group transfer. • There are two requirements for DNA polymerization: – All DNA Polymerases P l require i a template. t l t – All DNA Polymerases require a primer with a free 3’ – OH group for polymerization. Most primers are RNA that is synthesized by specialized enzymes (e.g. DNA Primase)

• The average number of nucleotides added before a DNA polymerase dissociates from a growing strand defines its processivity • DNA Polymerases vary greatly in this value. Chapter 25

Chapter 25

22

• Replication is VERY accurate. Good thing! • The pairing of bases depends on more than the hydrogen bonding patterns between the bases. • In addition, the active site of DNA polymerase accommodates only base pairs with aligned geometries. • An incorrect dNTP may be able to hydrogen bond with another base, but it generally will not fit in the enzyme active site! Chapter 25

24

6

DNA Replication

DNA Replication

DNA Polymerase

So, let’s replicate!

• Fit in the active site cannot account wholly for the high fidelity of replication • An incorrect base is still incorporated for every 104 to 105 bases added. • Almost all DNA polymerases have an intrinsic, separate 3’ → 5’ exonuclease activity that double-checks each dNTP after it is added. • This activity, known as proofreading, is not simply the reverse of the polymerization reaction. • Taken with the base selection, DNA Polymerase leaves behind one net error for every 106 to 108 bases added. Chapter 25

What needs to be done? (1) Locate the Origin of replication (2) Unwind the DNA (and continue to unwind it as we move along the template!) (3) We should also probably stabilize the ssDNA structure (4) Prime the DNA for the polymerase (remember PCR?) (5) Replicate the DNA (6) Replace the primer (if it was RNA!) (7) Fix the nicks (8) Find the terminus sequence and stop (9) CHECK OUR WORK! 25

Initiation

Elongation Termination 27

Chapter 25

DNA Replication

DNA Replication

DNA Polymerase

DNA Replicase System • Replication in E. coli requires 20 or more different enzymes and proteins, each performing a specific task. • Taken together, the entire complex is called the DNA Replicase System or the DNA Replisome • Components include:

DNA Polymerase III

– Helicases – moves along the DNA template and separates the strands using chemical energy (ATP) – Topoisomerases – relieves topological stress induced by strand separation. – DNA-Binding proteins – stabilize the separated DNA strands – Primases – synthesize the RNA primers on the template – DNA ligases – seal nicks in the phosphodiester backbone Chapter 25

26

Chapter 25

28

7

DNA Replication

DNA Replication

Initiation

Elongation • The replisome promotes rapid DNA synthesis, adding ~ 1000 nucleotides per second to each strand. • Once the Okazaki fragment has be completed, its RNA primer is removed and replaced with DNA by DNA Pol I • The remaining nick is sealed by DNA Ligase

Chapter 25

Chapter 25

29

Chapter 25

31

DNA Replication

DNA Replication

Elongation

DNA Ligase

30

Chapter 25

32

8

DNA Replication

DNA Repair

Termination

Mutations

• Eventally, replication forks will meet at a terminus region containing multiple copies of a 20 bp sequence called Ter • The Ter sequence is a binding site for the Tus protein (terminus utilization substance) and a Tus-Ter complex is formed. • When a replication fork runs into a Tus-Ter complex, it stops. • Replication halts when the second fork runs into the stopped fork Chapter 25

33

• Unrepaired DNA damage (a lesion) can result in a change in the base sequence of the DNA. • If replicated, this change can be passed onto daughter cells and become permanent, a mutation. • The mutation can involve base substitutions or the addition/ deletion of one or more base pairs. • If the mutation takes place in nonessential DNA or has a negligible effect on gene function, it is termed a silent mutation. • A typical mammalian genome accumulates 1000s of lesions in 24 hours • Our repair systems manage to keep mutations at around 1 in 1000 lesions. Chapter 25

35

DNA Replication

DNA Repair

Termination

Repair Systems

• Eventually, replication forks will meet at a terminus region containing multiple copies of a 20 bp sequence called Ter • The Ter sequence is a binding site for the Tus protein (terminus utilization substance) and a Tus-Ter complex is formed. • When a replication fork runs into a Tus-Ter complex, it stops. • Replication halts when the second fork runs into the stopped fork Chapter 25

34

Chapter 25

36

9

DNA Repair

DNA Repair

Mismatch Repair

Base-Excision Repair

• Mismatches are corrected to reflect the information of the original (template) strand. • The repair system must be able to discriminate between the new strand and the template. • In E. coli, the cell uses methylation of the template strand to “tag” it as the original strand. • This “tagging” is catalyzed by Dam methylase • Dam methylase methylates DNA at the N6 position of adenines within (5’)GATC sequences. • The “tagging” mechanism of other bacteria and eukaryotes is still unknown! Chapter 25

• The DNA Glycosylases recognize common DNA lesions, such as products of cytosine and adenine deamination, and remove the effected base by cleaving the N-glycosyl bond (CHP 8) • This cleavage leaves an apurinic or apyrimidinic site in the DNA (aka. The AP site or abasic b i site) it ) • Now, we need to repair the DNA. This is not merely attaching an undamaged base: – AP endonuclease cuts the backbone near the AP site, marking the lesion. – DNA Polymerse I removes a segment of DNA at the AP site then synthesizes a new strand from the free 3’ – OH. – DNA Ligase then seals the remaining nick. 37

Chapter 25

39

DNA Repair

DNA Repair

Mismatch Repair

Nucleotide-Excision Repair • DNA lesions can also cause large distortions of the helical structure of the dsDNA (Cyclobutane pyrimidine dimers for example) • These distortions are generally repaired by removing whole sections of DNA and replacing it with newly synthesized DNA (aka. Nucleotide - excision repair) Exinuclease because it can cleave at two sites within the same strand

Chapter 25

38

Chapter 25

40

10

DNA Repair

DNA Repair

Direct Repair

Recombinational DNA Repair

• Some damage can be repaired in place. • Once such process is catalyzed by the DNA Photolyases • These enzymes can utilize energy derived from absorbed light to reverse damage produced from UV light (Pyrimidine dimers!) • The chromophore of these enzymes is FADH- and a folate • Placental animals (including humans) do not have this class of enzymes. Chapter 25

41

•

A potentially dangerous type of DNA damage occurs when both strands of the double helix are broken, leaving no intact template strand for repair.

•

If these lesions were left unrepaired, they would quickly lead to the breakdown of chromosomes into smaller fragments

•

The end-joining mechanism, which can be viewed as an emergency solution to the repair of double-strand breaks, is a common outcome in mammalian cells. WHY is this OK??!!

•

The second mechanism transfers nucleotide sequence information from the intact DNA double helix of the homologous chromosome to the site of the double-strand break in the broken helix.

•

A DNA replication process uses the undamaged chromosome as the template for transferring genetic information to the broken chromosome, repairing it with no change in the DNA sequence Chapter 25

DNA Repair

Figure 5-53 Alberts et al. “Molecular Biology of the Cell” 4th Ed.

43

DNA Recombination

Direct Repair • Enzymes are also available to repair methylated bases. • The AlkB protein is an α-KG-Fe2+dependent dioxygenase

• The rearrangement of genetic information within and among DNA molecules can collectively be termed genetic recombination • These events can fall into three categories: – Homologous Genetic Recombination – the exchange of genetic information between any two DNA molecules (or segments) that share an extended region of nearly identical sequence. – Site-Specific Recombination – exchanges occur only at a particular DNA sequence. – DNA Transposition – usually involves a short segment of DNA with the capacity to move from one location in a chromosome to another.

• The function of these systems include: – DNA Repair; DNA Replication; Regulation of Gene Expression; Chromosome segregation; Maintenance of genetic diversity; and programmed genetic rearrangements during embryonic development. Chapter 25

42

Chapter 25

44

11

DNA Recombination

DNA Recombination

Homologous Genetic Recombination •

Transposons

The homologous genetic recombination reaction is essential for every proliferating cell because accidents occur in nearly every round of replication that interrupt the replication fork.

•

Recombination occurs with the highest frequency during meiosis

•

Recombination occurs due to the need to keep the two pairs of sister chromatids in close contact for even distribution among the resulting haploid gametes

•

Recombination also allows for the movement of transposable elements (aka. Transposons)

•

These segments of DNA are found in all cells and can “jump” from one place on a chromosome to another on the same or a different chromosome.

•

The new location is determined randomly, requiring tight regulation as insertion of an element into a vital gene could result in cell death death.

•

Movement is catalyzed by a Transposase that recognizes the terminal repeats at the end of a transposon. Once inserted into the new site, the overhangs are paired by DNA Polymerase and sealed by DNA Ligase.

•

• Chapter 25

45

This gives a short direct repeat of the sequence on either side of the insert Chapter 25

Figure 5-70 Alberts et al. “Molecular Biology of the Cell” 4th Ed.

47

Figure 5-55 Alberts et al. “Molecular Biology of the Cell” 4th Ed.

DNA Recombination

DNA Recombination

Site-Specific Recombination

Transposons - Immunoglobulins

•

Site-specific recombination is limited to specific sequences and is catalyzed by a system including enzymes and a unique recognition site

•

A DNA Recombinase recognizes a recombination site (20 to 200 bp sequence)

Chapter 25

• Transposition is an excellent way to introduce diversity into a specific gene area as long as it is regulated. • One example of programmed transposition is the generation of complete immunoglobulin genes from separate gene segments within the genome. • Like going to the iTunes (your genome) and putting together your own playlist (the final Ig) by selecting individual songs (the transposable gene segments). • Let’s us make millions of Igs without maintaining millions of separate genes. Cool! 46

Chapter 25

48

12

DNA Recombination Transposons - Retroviruses •

Outside the cell, a retrovirus (like HIV) exists as a singlestranded RNA genome packed into a protein capsid along with a virus-encoded reverse transcriptase enzyme.

•

Specific DNA sequences near the two ends of the doublestranded DNA product produced by reverse transcriptase are then th held together by a virus-encoded Figures 5-73 and 5-75 Alberts et al. “Molecular Biology of the Cell” 4 Ed. integrase enzyme. This integrase creates activated 3′-OH viral DNA ends that can directly attack a target DNA molecule through a mechanism very similar to that used by the cut-and-paste transposons.

•

Chapter 25

49

13