Chapter11DNA Replication and Synthesis

Semiconservative replication of DNA makes genetics continuity between parental and progeny cells possible, as predicted by Watson-Crick model.Each strand of the parent helix serves as a template for its complement. DNA synthesis is a complex but orderly process orchestrated by a myriad of enzyme and other molecules. Together, they function with great fidelity to polymerize nucleotides into polynucleotide chains. Other enzymes interact with DNA, leading to genetic recombination.

Key topics in this chapter

11.1 The mode of DNA replication

11.2 synthesis of DNA in microorganisms

11.3 DNA synthesis : A model

11.4 A coherent mode of DNA synthesis

11.5 Genetic control of replication

11.6 Eukaryotic DNA synthesis

11.7 DNA replication ,telomeres and telomerase

11.8 DNA recombination

1. The model of DNA Replication

DNA replication is semi-conservative, one strand serves as the template for the second strand. Furthermore, DNA replication only occurs at a specific step in the cell cycle. The following table describes the cell cycle for a hypothetical cell with a 24 hr cycle

Stage Activity Duration

G1 Growth and increase in cell size 10 hr

S DNA synthesis 8 hr

G2 Post-DNA synthesis 5 hr

M Mitosis 1 hr

DNA replication has two requirements that must be met:

1.DNA template 2.Free 3' -OH group

The Meselson-Stahl Experiment

• In 1958, Matthew Meselson and Franklin Stahl published the results of an experiment providing strong evidence that cells use semiconservative replication to produce new DNA molecules. Escherichia coli cells were grown for many generations in a medium where I5NH4C1 (ammonium chloride) was the only nitrogen source. A "heavy" isotope of nitrogen, I5N contains one more neutron than the naturally occurring 14N isotope. Unlike "radioactive" isotopes, 15N is stable and thus does not decay (i.e., it is not radioactive).

•After many generations, all nitrogen-containing molecules, including the nitrogenous bases of DNA, contained the heavier isotope in the E, coli cells. DNA containing I5N can be distinguished from '4N-containing DNA by the use of.

sedimentation equilibrium centrifugation, in which centrifugation "forces" samples through a density gradient of a heavy metal salt such as cesium chloride.

The more dense 15N-DNA reaches equilibrium in the gradient at a point closer to the bottom (where the density is greater) than14N-DNA

Semiconservative Replication in Eukaryotes

• In 1957, the year before the work of Meselson and his colleagues was published, J. Herbert Taylor, Philip Woods, and Walter Hughes presented evidence that semiconservative replication also occurs in eukaryotic organisms. They experimented with root tips of the broad bean Vicia faba, which are an excellent source of dividing cells. These researchers examined the chromosomes of these cells following replication of DNA. They monitored the replication process by labeling DNA with 3H-thymidine, a radioactive precursor of DNA, and then performing autoradiography.

• Autoradiography is a cytological technique that pinpoints the location of an isotope in a cell. In this procedure, a photographic emulsion is placed over a section of cellular material (root tips in this experiment), and the preparation is stored in the dark. The slide is then developed, much as photographic film is processed. Because the radioisotope emits energy, the emulsion turns black at the approximate point of emission. The end result is the presence of dark spots or "grains" on the surface of the section, locating the newly synthesized DNA in the cell.

Origins, Forks, and Units of Replication

• origin of replication. • Where along the chromosome is DNA replication

initiated? • Is there only a single origin, or does DNA synthesis

begin at more than one point? • Is a point of origin random or is it located at a

specific region along the chromosome? • Second, once replication begins, does it proceed in a

single direction or in both directions away from the origin? In other words, is replication unidirectional or bidirectional?

• replication fork

• Replicon the length of DNA that is replicated following one initiation event at a single origin is a unit called the,

In E. coli this specific region, called oriC, has been mapped along the chromosome. It consists of 245 base pairs, though only a small number are essential to the initiation of DNA synthesis.

3 9mers

4 11mers

Since in bacteriophages and bacteria DNA synthesis originates at a single point, the entire chromosome constitutes one replicon. The presence of only a single origin is characteristic of bacteria, which have only one circular chromosome.

11.2 synthesis of DNA in microorganisms

Proteins and enzymes of DNA Replication• DNA exists in the nucleus as a condensed,

compact structure. To prepare DNA for replication, a series of proteins aid in the unwinding and separation of the double-stranded DNA molecule. These proteins are required because DNA must be single-stranded before replication can proceed.

1. DNA Helicases - These proteins bind to the double stranded DNA and stimulate the separation of the two strands.

2. DNA single-stranded binding proteins - These proteins bind to the DNA as a tetramer and stabilize the single-stranded structure that is generated by the action of the helicases. Replication is 100 times faster when these proteins are attached to the single-stranded DNA.

3. DNA Gyrase - This enzyme catalyzes the formation of negative supercoils that is thought to aid with the unwinding process. In addition to these proteins, several other enzymes are involved in bacterial DNA replication.

4. Primase - The requirement for a free 3' hydroxyl group is fulfilled by the RNA primers that are synthesized at the initiation sites by these enzymes.

5. DNA Ligase - Nicks occur in the developing molecule because the RNA primer is removed and synthesis proceeds in a discontinuous manner on the lagging strand. The final replication product does not have any nicks because DNA ligase forms a covalent phosphodiester linkage between 3'-hydroxyl and 5'-phosphate groups.

6. DNA Polymerase - DNA Polymerase I (Pol I) was the first enzyme discovered with polymerase activity, and it is the best characterized enzyme. Although this was the first enzyme to be discovered that had the required polymerase activities, it is not the primary enzyme involved with bacterial DNA replication. That enzyme is DNA Polymerase III (Pol III). Three activities are associated with DNA polymerase I;

• 5' to 3' elongation (polymerase activity) • 3' to 5' exonuclease (proof-reading activity) • 5' to 3' exonuclease (repair activity) The second two activities of DNA Pol I are importan

t for replication, but DNA Polymerase III (Pol III) is the enzyme that performs the 5'-3' polymerase function.

DNA Polymerases II and III

• Although DNA synthesized under the direction of polymerase I demonstrated biological activity, a more serious reservation about the enzyme's true biological role was raised in 1969. Peter DeLucia and John Cairns reported the discovery of a mutant strain of E. coli that was deficient in polymerase I activity. The mutation was designated polA1. In the absence of the functional enzyme, this mutant strain of E. coli still duplicated its DNA and reproduced successfully! Other properties of the mutation led DeLucia and Cairns to conclude that in the absence of polymerase I, these cells are highly deficient in their ability to "repair" DNA.

• For example, the mutant strain is highly sensitive to ultraviolet light and radiation, both of which damage DNA and are therefore mutagenic. Non mutant bacteria are able to repair a great deal of UV-induced damage. These observations led to two conclusions:

• 1. At least one other enzyme that is responsible for replicating DNA in vivo is present in E. coli cells.

• 2. DNA polymerase I may serve a secondary function in vivo. This function is now believed by Komberg and others to be critical to the fidelity of DNA synthesis,but this enzyme does not actually synthesize the entire complementary strand during replication.

Table 11.2 Properties of Bacterial DNA Pol I, II andIII

Properties I II III

Initiation - - -

5’-3’ Polymerization + + +

3’-5’ exonuclease activity + + +

5’-3’ exonuclease activity + - -

Molecules of pol/cell 400 ? 15

Gene Function

dnaA,I,P Initiation

dnaB,C Helicase at oriC

dnaE,N,Q,X,Z Subunits of DNA polymerase III

dnaG Primase

gyrA,B Subunits of gyrase

lig Ligase

oriC Origin of Replication

polA DNA polymerase I

polB DNA polymerase II

rep Helicase

ssb Single-stranded DNA binding proteins

• 11.3 DNA Synthesis :A General Model for DNA Replication1. The DNA molecule is unwound and prepared for

synthesis by the action of DNA gyrase, DNA helicase and the single-stranded DNA binding proteins.

2. A free 3‘-OH group is required for replication, but when the two chains separate no group of that nature exists. RNA primers are synthesized, and the free 3'OH of the primer is used to begin replication.

3. The replication fork moves in one direction, but DNA replication only goes in the 5' to 3' direction. This paradox is resolved by the use of Okazaki fragments.

Unwinding the DNA helix

This region of the E. coii chromosome has been particularly well studied. Called oriC, it consists of 245 base pairs characterized by repeating sequences of 9 and 13 bases (called 9mers and 13mers). One particular protein (called DnaA because it is encoded by the gene dnaA) initiates unwinding of the helix. A number of subunits of the DnaA protein bind to each of several 9mers. This step is essential in facilitating the subsequent binding of DnaB and DnaC proteins that further open and destabilize the helix (Figure 11-9). Proteins such as these, which require the energy normally supplied by ATP hydrolysis to break hydrogen bonds and denature the double helix, are called helicases. Other proteins, called single-stranded binding proteins (SSBPs) stabilize

4.These are short, discontinuous replication products that are produced off the lagging strand. This is in comparison to the continuous strand that is made off the leading strand.

5.The final product does not have RNA stretches in it. These are removed by the 5' to 3' exonuclease action of Polymerase I.

6.The final product does not have any gaps in the DNA that result from the removal of the RNA primer. These are filled in by the action of DNA Polymerase I.

7. DNA polymerase does not have the ability to form the final bond. This is done by the enzyme DNA ligase.

Initiation of DNA synthesis

Continuous and Discontuous DNA synthesis

Concurrent synthesis on the leading and lagging strands

Proofreading and error correction during DNA replication

11.6 Eukaryotic DNA Synthesis

• Research shows that eukaryotic DNA is replicated in a man ner similar to tha£ of bacteria. Eukaryotic polymerases have the same fundamental requirements for DNA synthesis as do bacteri al systems; four deoxyribonucleoside triphosphates, a tem plate, and a primer. However, because eukaryotic cells contain much more DNA per cell and because this DNA is complexed with proteins, eukaryotes face many problems not encountered by bacteria. As we might expect, these com plications make the process of DNA synthesis much more complex in eukaryotes and more difficult to study. However, a great deal is now known about the process.

Multiple Replication OriginsMultiple Replication Origins

Multiple Replication Origins

The most obvious difference between eukaryotic and prokaryotic DNA replication is that eukaryotic chromosomes contain multiple replication origins, in contrast to the single site that is part of the E. coli chromosome.

The multiple origins are visible under the electron microscope (Figure 11-14).

• They are essential if the entire genome of a typical eukaryote is to be replicated in a reasonable time. Recall that (1) eukaryotes have much greater amounts of DNA than bacteria (e.g., yeast has 4 times as much and Drosophila has 100 times as much DNA as E. coli); and (2) the rate of synthesis by eukaryotic DNA polymerase is much slower—only about 50 nucleotides per second, a rate 20 times less than the comparable bacterial enzyme. Under these conditions, single-origin replication of a typical eukaryotic genome would take up to one month to complete! However, replication is accomplished in as little as 3 minutes in some eukaryotic organisms.

• Yeast 250-400 replicon, mammalian 2500

autonomously replicating sequences (ARSs). a unit of 11 base pairs, flanked by other short sequences involved in efficient initiation. DNA synthesis is restricted to the S phase of the eukaryotic cell cycle. Research has shown that the many origins are not all activated at once; instead clusters of 20-80 adjacent replicons are activated sequentially throughout the S phase until all DNA is replicated.

Eukaryotic DNA Polymerases

• The most complex aspect of eukaryotic replication is the array of polymerases involved in directing DNA synthesis. A total of six different forms of the enzyme have been isolated and studied. For the polymerases to access DNA, the topology of the helix must first be modified. As synthesis is triggered at each origin site, the double strands are opened up in an A = T-rich region, which allows a helicase enzyme to enter that further unwinds the double-stranded DNA. before polymerases begin synthesis, histone proteins complexed to the DNA also must be stripped away or otherwise modified.

• As DNA synthesis then proceeds, histones reassociate with the newly formed duplexes, reestablishing the characteristic nucleosome pattern Figure 11-15). In eukaryotes, the synthesis of new histone proteins is tightly coupled to DNA synthesis during the S phase of the cell cycle.

•

11.7 DMA Replication, Telomeres, and Telomerase

• A final difference that exists between prokaryotic and eukaryotic DNA synthesis involves the nature of the chromosomes. Unlike the closed, circular DNA of bacteria and most bacteriophages, eukaryotic chromosomes are linear. During replication they face a special problem at the "ends" of these linear molecules, called the telomeres. While synthesis proceeds normally to the end of the leading strand, a difficulty arises on the lagging strand as the RNA primer is removed (Figure 11-16).

• Normally, the newly created gap would be filled by adding a nucleotide to the existing 3'-OH group provided during discontinuous synthesis (to the right of gap b in Figure 11-16). However, there is no strand present to provide the 3 '-OH group because this is the end of the chromosome. As a result, each successive round of synthesis theoretically shortens the chromosome by the length of the RNA primer. Because this is such a significant problem, we can suppose, at least for some cells, that a molecular solution would have developed early in evolution and be shared by all

Centromeres and Telomeres

Telomeres - the region of DNA at the end of linear eukaryotic chromosome; required for the replication and stability of the chromosome

McClintock recognized several features about the ends of chromosomes • If two chromosomes were broken in a cell, the end of one c

ould attach to the other and vice versa • She never observed was the attachment of the broken end to

the end of an unbroken chromosome. • Thus the ends of broken chromosomes are sticky, whereas t

he normal end is not sticky, • This suggests the ends of chromosomes have unique feature

s. • Usually, but not always, the telomeric DNA is heterochrom

atic and contains direct tandemly repeated sequences; the sequence TTAGGG is often the tandemly repeated sequence.

Telomere Repeat Sequences

Species Repeat Sequence

Arabidopsis TTTAGGG

Human TTAGGG

Oxytricha TTTTGGGG

Slime Mold TAGGG

Tetrahymena TTGGGG

Trypanosome TAGGG

Yeast (TG)1-3TG2-3

Replication of Telomeres •Because DNA synthesis requires an RNA template (that provides the free 3'-OH group) to prime the synthesis, then a few bases at the end of a lagging strand would not be replicated during each round of replication •This would shorten the length of the chromosome after each division.  But this is not seen. •Apparently telomerases, enzymes that replicate the terminal end of the chromosome exist. •These enzymes have a mode of action different than a normal DNA polymerase, but the end result is that sequences at the end of the chromosome do not go unreplicated during each replication round.

11.8 DNA Recombination

• We conclude this chapter by returning to a topic discussed in Chapter 8—genetic recombination. There, we pointed out that the process of crossing over depends on breakage and rejoining of the DNA strands between homologs. Now that we have discussed the chemistry and replication of DNA.. it is appropriate to consider how recombination occurs at the molecular level. In general, the following information pertains to genetic exchange between any two homologous double-stranded DNA molecules, whether they be viral or bacterial chromosomes or eukaryotic homologs during Genetic exchange at equivalent positions along two chromosomes with substantial DNA sequence homology is referred to as general or homoloeous recombination.

• Several models attempt to explain crossing over, and they all share certain common features. First, all are based on the initial proposals put forth independently by Robin Holliday and Harold L. K. Whitehouse in 1964. They also depend on the complementarity between DNA strands for their precision of exchange. Finally, each model relies on a series of enzymatic processes to accomplish genetic recombination.

• One such model is shown in Figure 11-18. It begins with two paired DNA duplexes or homologs

Gene Conversion

• A modification of the preceding model has helped us better understand a unique genetic phenomenon known as gene conversion( 基因转变） .

• Initially found in yeast by Carl Lindegren and in Neurospora by Mary Mitchell, gene conversion is characterized by a genetic exchange ratio involving two closely linked genes that is nonreciprocal. If we cross two Neurospora strains each bearing a separate mutation (a+ X b+), a reciprocal recombination event between the genes yields spore pairs of the + + and ab genotypes. However, a nonreciprocal exchange yields one pair without the other.

• Working with pyridoxine mutants, Mitchell observed several asci containing spore patterns displaying the ++ genotype, but not the reciprocal product (ab). Because the frequency of these events was higher than the predicted mutation rate and thus could not be accounted for by that phenomenon, they were called "gene conversions." They were so named because it appeared that one allele had somehow been "convened" to another during an event in which genetic exchange also occurred. Similar findings are apparent in the study of other fungi as well.

•Gene conversion is now considered to be a consequence of the recombination process. One possible explanation interprets conversion as a mismatch of base pairs during heteroduplex formation, as shown in Figure 11—19. Mismatched regions of hybrid strands can be repaired by excising one

基因转变与异常分离 （遗传学 第 8 ） p198 基因转变 Gene conversion 及其分子机制

• 通常认为，重组总是交互的。例 1 ：脉孢菌：杂合体 Aa ，一个染色体把基因

A 交给它的同源染色体，同源染色体必然把基因 a 返回来叫给它。所以真菌中的一个位点（ locus ）上两个等位基因的分离应呈 2 ： 2 分离。

• 但是在脉孢菌中发现，有的子囊含有（ 3A+1a ）或（ 1A+3a ）的子囊孢子。

• Mitchell 对这种现象进行了详细的分析。他的杂交实验中用的是吡哆醇合成的基因。

有两个突变位点，接近：一个位点上：一个突变基因 pdxp—— 突变株

在吡哆醇培养基上生长，对 pH 敏感，变酸后则不用再添加了。

相邻的一个位点：也有突变基因，也是吡哆醇需要型——但对 pH 不敏感 pdx

+pdxp x pdx + 杂交 对 585 个子囊孢子培养鉴定。结果：

在 4 个子囊中有野生型孢子对——好象两者之间发生了重组

但是，与预期的重组结果不同——没有出现双突变型（ pdxp pdx ）孢子对（图 12-13 ）。

• 应该说分析结果是灵敏度，只要有双突变型应该能检出的。

• 这种情况也不能用突变说明，因为出现的频率比突变率高得多。

• 这种情况好象由一个基因转变为另一等位基因（与突变的区别是什么）——所以被称为基因转变（ gene conversion ）， pdxp 转变为 pdxp+ （或 + ）。

• 基因转变往往伴随有转换区外基因的重组，区外基因重组是交互的，所以 pdxp 位点出现异常的 3 ： 1 或 6 ： 2 分离，但 pdx 位点仍显示正常的 2 ： 2 分离。

例 2 ：• 粪生粪壳菌，基因转变在粪生粪壳菌中也有发现。

在该菌中，有一个基因座位 g 。 Olive 进行了广泛的研究

• g-—— 决定子囊孢子灰色• g+—— 决定子囊孢子黑色 g+ X g- 杂交——分析

了 20 万个子囊。• 结果发现： 5 ： 3 分离的占 0.06%• 6 : 2 0.05%• 3:1:1:3 ( 异常 4 ： 4) 分离的占 0.008%

（图 12-14 ）• 另外，在该菌中，虽然 g/+ 这对等位基因表现许多异

常分离，但其邻近的基因 A/a 都呈现正常分离。与前面面包酵母一样（图 12-15 ）

基因转变的类型• 基因转变有如下几种类型（图8-6）：

l ．染色单体转变（ chromatid conversion ）减数分裂的 4 个产物中，有一个产物发生了基因转变，所以称染色单体转变，出现 6 ： 2 （或 2:6 ）的子囊。如图8-6中的 A 所示。

2 ．半染色单体转变（ half － chromatid conversion ）5:3 （或 3:5 ）或 3:1:1:3 的子囊则表明在减数分裂的4 个产物中，有一个产物的一半或两个产物的各一半出现基因转变，称半染色单体转变。因为在 5:3 。 3:1:1:3 的分离中，基因转变只影响半个染色单体，分离一定发生在减数分裂后的有丝分裂中，所以又称减数后分离（ post-meiotic segregation ）。如图8-6中 B 、 C 所示。基因转变也在子囊菌 Ascobolus immersus 和果蝇（ D ． virilis ）中发现，在 D ． virilis 中的 re 基因转变为 re+ ，同时其旁侧基因 y 与 sc 间的交换率提高了 24 倍。

• 在 6 ： 2 （ 2 ： 6 ）分离的子囊中，减数分裂 4 个产物，有一个基因转变——染色单体转变。

• 而在 5 ： 3 （ 3 ： 5 ）和 3 ： 1 ： 1 ： 3 的子囊中， 4 个产物有一个的一半出现基因转变——为半染色体转变，也就是说影响半个染色单体，所以分离一定发生在减数分裂后的有丝分裂——叫减数后分离。

同源重组的 Holliday 模型

• 断裂愈合学说 : 1937 年由 Darlington, 认为两条染色单体在相对应的位点上断裂然后重新连接起来。

• 从分子水平来说， DNA 分子在同一碱基对间断裂，然后以新的组合连接起来。

• 如果染色单体不是在相应的位点断裂和融合，那么就可能造成染色体的重复或缺失。但这种情况自然是很少见的。

• 四分子分析，使我们对重组现象有了进一步的认识：• ①     重组有正常的交互方式• ②     偶然也有不规则的非交互方式，这种非交互式

则是基因转变• ③     基因转变往往与正常互换有关，两者都涉及相

同的两条非姊妹染色单体• 有的子囊可表现为减数后分离，重组可影响染色单体，

也可影响半染色单体。 • 为了解释这些现象或者说阐明这些事实，学者们提出

了不同的重组模型，其中 Holliday 提出的“异源或杂种 DNA 模型 heteroduplex”受到多数人的支持。这个模型又称为 Holliday 模型，该模型既适用于原核也适用于真核生物。那么，这种模型是什么

a、同源非姊妹染色单体联会

b、 DNA内切酶相同位置切开

c、交换重接

d、形成交联桥结构

e、交联桥沿DNA分子移动，产生一大段异源双链——称为 Holliday结构

a、交联桥旋转 180度

b、 Holliday结构异构体

c、两种方式切开

c 和 d 结构相同，如左右切断形成的线性分子是非重组体（ AB 、 ab ），如上下切断形成的线性分子其异源双链 DNA 区的两侧标记基因是重组体（ Ab 、 aB ）。由此可知，不管 Holliday 结构断裂是否导致旁侧遗传标记的重组，它们都含有一个异源双链DNA 区（由 G-C 、 A-T配对变为 G-A 、 C-T非配对）。

• 两个方向 东西断开——仍得亲代类型 AB ab南北断开——重组类型 Ab aB但无论那种断开， A-B 两个座位间都有一

DNA 区段是异源双链。

– 因为异源双链有关的两个核苷酸链分别来自不同亲体，如果杂交的两个亲体为 g+×g- ，假设两者有一对碱基之差，

g+( 或 +)

g-( 或 -)

• 异源双链 DNA 区域应为 带有不相

对称碱基对的 DNA 不稳定，必将修补。• 如何修补，修补可带来什么样的结果？

ACAGT

TGTCA

ACATT

TGTAAACAGT

TGTAA

• 该模型认为：（ 1 ）不对称核苷酸由核酸外切酶切除，留下缺口。（ 2 ）再由 DNA聚合酶作用，合成具有互补的碱基

区段填补缺口。（ 3 ）然后再由连接酶作用，连接• 这个修补过程可能有两种情况。例如不相称碱基

对 G-A 的修补： G （ =+ ）—— A G C 野生型 +A （ =g ）—— G T A 突变型 g如果不相称的核苷酸没有得到校正，杂种 DNA （杂

种染色体）留到下一次复制，将产生不同的子染色单体——这样就出现了半染色单体转变。一个孢子对中两个孢子不同（图 12-18 ）

1．两个杂种分子均未校正（图8-8A）复制后出现异常的 4+:4g（或 3:1:1:3）的分离。由于不配对的减基没有得到修复校正，杂种 DNA留到下一次复制。在下一次复制即减数分裂以后的复制中，异源双链DNA复制形成两个不同的等位基因，通过有丝分裂发生分离。减数分裂的 4个产物中的两个染色单体的各 1 ／ 2出现基因转变，即仅仅影响 DNA双链中的一条链，实际只影响两个单体中的半个染色单体，即DNA双链中的各一条链。显然转变的链与未转变的链的分离一定是在减数分裂后的有丝分裂中发生。因而这类基因转变属于半染色单体转变，或减数后分离，子囊孢子的异常分离比为 3:1:1:3

2．一个杂种分子校正为 +，或校正为 g时，则发生另一种类型的半染色单体转变，前者修复后出现 5:3的分离，后者于囊抱子的异常分离比为 3:5（图8-8B）。由图 8—8B可见，减数分裂 4个产物中的一个产物（第3条染色单体或第2条染色单体）的 1／2出现转变。由于只影响半个染色单体，所以转变的链与未转变的链将在减数分裂后的有丝分裂中发生分离。 综合上述两种修复情况可见，半染色单体转变是当两个杂种分子都未进行修复校正或一个杂种分子校正到+（或 g）时，使得减数分裂的 4个产物中的两个产物的各 1/2或一个产物的 1／ 2发生了基因转变。其子囊抱子的典型异常分离比例分别为 3： 1： 1： 3以及 5:3或 3:5。

3．两个杂种分子都被校正到+（或 g）时（图 8－8C），修复后出现 6＋： 2g（或 2＋： 6g）的异常分离。这便是出现染色单体转变的起因。 4．当两个杂种分子都按原来两个亲本的遗传结构进行修复时，则减数分裂 4个产物恢复成 G－ C、G－ C、 A－ T、 A－ T的正常配对状态，子囊抱子分离正常，呈现 4+： 4g的结果，如图 8－ 8D所示。 切除修复系统识别异源双链DNA中错配的碱基对时，切除其中的一条链，使其恢复互补性。于是，使得代表一个等位基因的 DNA链变成了代表另一个等位基因的序列。如果这种校正变化只发生在一个异源双链DNA上，就会产生 5： 3（或 3:5）异常分离。如果两个异源双链以同样方式校正，就产生了 2： 6（或 6： 2）异常分离。所以基因转变实质上是异源双链DNA错配的核苷酸对在修复校正过程中所发生的一个基因转变为它的等位基因的现象。

• 同样，杂合双链 +/g校正为 g 的情况应该也是如此。换言之，第 2 个分子校正为 +还是第 3 个分子校正为 + 的机会应相同。对于 8 个子囊孢子的子囊来讲， 5+ ： 3g的情况就是连续排列的 +++++ggg；而四型子囊则是不连续的 +++g++gg排列。 实际上，三型子囊的数目经常显著的多于四型。根据 1982 年 Whitehouse 所收集的一组数据可以说明（表 8-2 ）。

• (a), each of which has a single-stranded nick introduced

• (b) at an identical position by an endonuclease. The ends of the strands produced by these cuts are then displaced and subsequently pair with their complements on the opposite duplex

• (c). A ligase then seals the loose ends • (d), creating hybrid duplexes called heteroduplex DNA

molecules. The exchange creates a cross-bridged or Holliday structure. The position of the cross bridge then moves down the chromosome as a result of a process called branch migration

•

• (e). This occurs as a result of a zip-perlike action as hydrogen bonds break and then reform between complementary bases of the displaced strands of each duplex. This migration yields an increased length of heteroduplex DNA on both homologs.

• If the duplexes separate • (f) and the bottom portions rotate 180° • (g), an intermediate planar structure called a chi

form is created. If the two strands on opposite homologs previously uninvolved in the exchange are now nicked by an endonuclease

• (h) and ligation occurs • (i), recombinant duplexes are created. Note that

the arrangement of alleles is altered as a result of recombination.

• Evidence supporting this model includes the electron microscopic visualization of chi-form planar molecules from bacteria where four duplex arms join at a single point of exchange [Figure 1 l-18(g)]. Further important evidence comes from the discovery in E. coli of the RecA protein. This molecule promotes the exchange of reciprocal single-stranded DNA molecules as occurs in step (c) of the model..

• RecA also enhances the hydrogen-bond formation during strand displacement, thus initiating heteroduplex formation. Finally, many other enzymes essential to the nicking and ligation process have also been discovered and investigated. The products of the recB, recC, and recD genes are thought to be involved in the nicking and unwinding of DNA. Numerous mutations that prevent genetic recombination have been found in viruses and bacteria. These mutations are thought to identify genes, whose products play an essential role in this process

End up here,see you next• Eukaryotic Chromosome Structure • The length of DNA in the nucleus is far greater than the size of the compartment in which it is contained. To fit into this compartment the DNA has to be condensed in some manner. The degree to which DNA is condensed is expressed as its packing ratio. • Packing ratio - the length of DNA divided by the length into which it is packaged • For example, the shortest human chromosome contains 4.6 x 107 bp of DNA (about 10 times the genome size of E. coli). This is equivalent to 14,000 µm of extended DNA. In its most condensed state during mitosis, the chromosome is about 2 µm long. This gives a packing ratio of 7000 (14,000/2). • To achieve the overall packing ratio, DNA is not packaged directly into final structure of chromatin. Instead, it contains several hierarchies of organization. The first level of packing is achieved by the winding of DNA around a protein core to produce a "bead-like" structure called a nucleosome. This gives a packing ratio of about 6. This structure is invariant in both the euchromatin and heterochromatin of all chromosomes. The second level of packing is the coiling of beads in a helical structure called the 30 nm fiber that is found in both interphase chromati

n and mitotic chromosomes. This structure increases the packing ratio to about 40. The final packaging occurs when the fiber is organized in loops, scaffolds and domains that give a final packing ratio of about 1000 in interphase chromosomes and about 10,000 in mitotic chromosomes. • Eukaryotic chromosomes consist of a DNA-protein complex that is organized in a compact manner which permits the large amount of DNA to be stored in the nucleus of the cell. The subunit designation of the chromosome is chromatin. The fundamental unit of chromatin is the nucleosome. • Chromatin - the unit of analysis of the chromosome; chromatin reflects the general structure of the chromosome but is not unique to any particular chromosome • Nucleosome - simplest packaging structure of DNA that is found in all eukaryotic chromosomes; DNA is wrapped around an octamer of small basic proteins called histones; 146 bp is wrapped around the core and the remaining bases link to the next nucleosome; this structure causes negative supercoiling • The nucleosome consists of about 200 bp wrapped around a histone octamer that contains two copies of histone proteins H2A, H2B, H3 and H4. These are known as the core histones. Histones are basic proteins that have an affinity for DNA and are the most abundant proteins associated with DNA. The amino acid sequence of these four histones is conserved suggesting a similar function for all. • The length of DNA that is associated with the nucleosome unit varies between species. But regardless of the size, two DNA components are involved. Core DNA is the DNA that is actually associated with the histone octamer. This value is invariant and is 146 base pairs. The core DNA forms two loops around the octamer, and this permits two regions that are 80 bp apart to be brought into close proximity. Thus, two sequences that are far apart can interact with the same regulatory protein to control gene expression. The DNA that is between each histone

octamer is called the linker DNA and can vary in length from 8 to 114 base pairs. This variation is species specific, but variation in linker DNA length has also been associated with the developmental stage of the organism or specific regions of the genome. •

• The next level of organization of the chromatin is the 30 nm fiber. This appears to be a solenoid structure with about 6 nucleosomes per turn. This gives a packing ratio of 40, which means that every 1 µm along the axis contains 40 µm of DNA. The stability of this structure requires the presence of the last member of the histone gene family, histone H1. Because experiments that strip H1 from chromatin maintain the nucleosome, but not the 30 nm structure, it was concluded that H1 is important for the stabilization of the 30 nm structure. • The final level of packaging is characterized by the 700 nm structure seen in the metaphase chromosome. The condensed piece of chromatin has a characteristic scaffolding structure that can be detected in metaphase chromosomes. This appears to be the result of extensive looping of the DNA in the chromosome. • The last definitions that need to be presented are euchromatin and heterochromatin. When chromosomes are stained with dyes, they appear to have alternating lightly and darkly stained regions. The lightly-stained regions are euchromatin and contain single-copy, genetically-active DNA. The darkly-stained regions are heterochromatin and contain repetitive sequences that are genetically inactive. • Centromeres and Telomeres • Centromeres and telomeres are two essential features of all eukaryotic chromosomes. Each provide a unique function that is absolutely necessary for the stability of the chromosome. Centromeres are required for the segregation of the centromere during me iosis and mitosis, and teleomeres provide terminal stability to the chromosome and ensure its survival. • Centromeres are those condensed regions within the chromosome that are responsible for the accurate segregation of the replicated chromosome during mitosis and meiosis. When chromosomes are stained they typically show a dark-stained region that is the centromere. During mitosis, the centromere that is shared by the sister chromatids must divide so that the chromatids can migrate to opposite poles of the cell. On the other hand, during the first meiotic division the centromere of sister chromatids must remain intact, whereas during meiosis II they

must act as they do during mitosis. Therefore the centromere is an important component of chromosome structure and segregation. • Within the centromere region, most species have several locations where spindle fibers attach, and these sites consist of DNA as well as protein. The actual location where the attachment occurs is called the kinetochore and is composed of both DNA and protein. The DNA sequence within these regions is called CEN DNA. Because CEN DNA can be moved from one chromosome to another and still provide the chromosome with the ability to segregate, these sequences must not provide any other function. • Typically CEN DNA is about 120 base pairs long and consists of several sub-domains, CDE-I, CDE-II and CDE-III. Mutations in the first two sub-domains have no effect upon segregation, but a point mutation in the CDE-III sub-domain com pletely eliminates the ability of the centromere to function during chromosome segregation. Therefore CDE-III must be actively involved in the binding of the spindle fibers to the centromere. • The protein component of the kinetochore is only now being characterized. A complex of three proteins called Cbf-III binds to normal CDE-III regions but can not bind to a CDE-III region with a point mutation that prevents mitotic segregation. Fur thermore, mutants of the genes encoding the Cbf-III proteins also eliminates the ability for chromosomes to segregate during mitosis. Additional analyses of the DNA and protein components of the centromere are necessary to fully understand the mechanics of chromosome segregation. • Telomeres are the region of DNA at the end of the linear eukaryotic chromosome that are required for the replication and stability of the chromosome. McClintock recognized their special features when she noticed, that if two chromosomes were broken in a cell, the end of one could attach to the other and vice versa. What she never observed was the attachment of the broken end to the end of an unbroken chromosome. Thus the ends of broken chromosomes are sticky, whereas the normal end is not sticky, suggesting the ends of chromosomes have u

nique features. Usually, but not always, the telomeric DNA is heterochromatic and contains direct tandemly repeated sequences. The following table shows the repeat sequences of several species. These are often of the form (T/A)xGy where x is between 1 and 4 and y is greater than 1. • Telomere Repeat Sequences • Species • Repeat Sequence• ArabidopsisTTTAGGG• Human• TTAGGG• OxytrichaTTTTGGGG• Slime Mold• TAGGG• TetrahymenaTTGGGG• Trypanosome• TAGGG• Yeast• (TG)1-3TG2-3

• Notice that the number of TG sequences and the number of cytosines in the yeast sequence varies. At least for yeast, it has been shown that different strains contain different lengths of teleomeres and that the length is under genetic control. • The primary difficulty with telomeres is the replication of the lagging strand. Because DNA synthesis requires a RNA template (that provides the free 3'-OH group) to prime DNA replication, and this template is eventually degraded, a short single-stranded region would be left at the end of the chromosome. This region would be susceptible to enzymes that degrade single-stranded DNA. The result would be that the length of the chromosome would be shortened after each division. But this is not seen. • The action of the telomerase enzymes ensure that the ends of the lagging strands are replicated correctly. A well-studied system involves the Tetrahymena protozoa organism. The telomeres of this organism end in the sequence 5'-TTGGGG-3'. T he telomerase adds a series of 5'-TTGGGG-3' repeats to the ends of the lagging strand. A hairpin occurs when unusual base pairs between guanine residues in the repeat form. Next the RNA primer is removed, and the 5' end of the lagging strand can be used for DNA synthesis. Ligation occurs between the fi

nished lagging strand and the hairpin. Finally, the hairpin is removed at the 5'-TTGGGG-3' repeat. Thus the end of the chromosome is faithfully replicated. The following figure shows these steps.

• The Replication of Telomeres• • Analysis of DNA Sequences in Eukaryotic Genomes • The technique that is used to determine the sequence complexity of any genome involves the denaturation and renaturation of DNA. DNA is denatured by heating which melts the H-bonds and renders the DNA single-stranded. If the DNA is rapidly cooled, the DNA remains single-stranded. But if the DNA is allowed to cool slowly, sequences that are complementary will find each other and eventually base pair again. The rate at which the DNA reanneals (another term for renature) is a function of the species from which the DNA was isolated. Below is a c

urve that is obtained from a simple genome. • The Y-axis is the percent of the DNA that remains single stranded. This is expressed as a ratio of the concentration of single-stranded DNA (C) to the total concentration of the starting DNA (Co). The X-axis is a log-scale of the product of the initial concentration of DNA (in moles/liter) multiplied by length of time the reaction proceeded (in seconds). The designation for this value is Cot and is called the "Cot" value. The curve itself is called a "Cot" curve. As can be seen the curve is rather smooth which indicates that reannealing occurs slowing but gradually

over a period of time. One particular value that is useful is Cot½ , the Cot value where half of the DNA has reannealed. • Steps Involved in DNA Denaturation and Renaturation Experiments • 1. Shear the DNA to a size of about 400 bp.

2. Denature the DNA by heating to 100oC.3. Slowly cool and take samples at different time intervals.4. Determine the % single-stranded DNA at each time point.

• The shape of a "Cot" curve for a given species is a function of two factors: 1. the size or complexity of the genome; and 2. the amount of repetitive DNA within the genome

• If we plot the "Cot" curves of the genome of three species such as bacteriophage lambda, E. coli and yeast we will see that they have the same shape, but the Cot½ of the yeast will be largest, E. coli next and lambda smallest. Physically, the larger the genome size the longer it will take for any one sequence to encounter its complementary sequence in the solution. This is because two complementary sequences must encounter each other before they can pair. The more complex the genome, that is the more unique sequences that are available, the longer it will take for any two complementary sequences to encounter each other and pair. Given similar concentrations in solution, it will then take a more complex species longer to reach Cot½ .

• • Repeated DNA sequences, DNA sequences that are found more than once in the genome of the species, have distinctive effects on "Cot" curves. If a specific sequence is represented twice in the genome it will have two complementary sequences to pair with and as such will have a Cot value half as large as a sequence represented only once in the genome. • Eukaryotic genomes actually have a wide array of sequences that are represented at different levels of repetition. Single copy sequences are found once or a few times in the genome. Many of the sequences which encode functional genes fall into this class. Middle repetitive DNA are found from 10s - 1000 times in the genome. Examples of these would include rRNA and tRNA genes and storage proteins in plants such as corn. Middle repetitive DNA can vary from 100-300 bp to 5000 bp and can be dispersed throughout the genome. The most a

bundant sequences are found in the highly repetitive DNA class. These sequences are found from 100,000 to 1 million times in the genome and can range in size from a few to several hundred bases in length. These sequences are found in regions of the chromosome such as heterochromatin, centromeres and telomeres and tend to be arranged as a tandem repeats. The following is an example of a tandemly repeated sequence: • ATTATA ATTATA ATTATA // ATTATA • Genomes that contain these different classes of sequences reanneal in a different manner than genomes with only single copy sequences. Instead of having a single smooth "Cot" curve, three distinct curves can be seen, each representing a different repetition class. The first sequences to reanneal are the highly repetitive sequences because so many copies of them exist in the genome, and because they have a low sequence complexity. The second portion of the genome to reanneal is the middle repetitive DNA, and the final portion to reanneal is the si

ngle copy DNA. The following diagram depicts the "Cot" curve for a "typical" eukaryotic genome •

• The following table gives the sequence distribution for selected species. • Species Sequence Distribution Bacteria 99.7% Single Copy Mouse 60% Single Copy

25% Middle Repetitive10% Highly Repetitive Human 70% Single Copy13% Middle Repetitive8% Highly Repetitive Cotton 61% Single Copy27% Middle Repetitive8% Highly Repetitive Corn 30% Single Copy40% Middle Repetitive20% Highly Repetitive Wheat 10% Single Copy83% Middle Repetitive4% Highly Repetitive Arabidopsis 55% Single Copy27% Middle Repetitive10% Highly Repetitive

• Sequence Interspersion • Even though the genomes of higher organisms contain single copy, middle repetitive and highly repetitive DNA sequences, these sequences are not arranged similarly in all species. The prominent arrangement is called short period interspersion. This arrangement is characterized by repeated sequences 100-200 bp in length interspersed among single copy sequences that are 1000-2000 bp in length. This arrangement is found in animals, fungi and plants. • The second type of arrangement is long-period interspersion. This is characterized by 5000 bp stretches of repeated sequences interspersed within regions of 35,000 bp of single copy DNA. Drosophila is an example of a species with this uncommon sequence arrangement. In both cases, the repeated sequences are usually from the middle repetitive class. We discussed above where highly repetitive sequences are found. • Eukaryotic Chromosome Karyotype • Whereas bacteria only have a single chromosome, eukaryotic species have at least one pair of chromosomes. Most have more than one pair. Another relevant point is that eukaryotic chromosomes are detected only occur during cell division and not during all stages of the cell cycle. They are in their most condensed form during metaphase when the sister chromatids are attached. This is the primary stage when cytogenetic analysis is performed. • Each species is characterized by a karyotype. The karyotype is a description of the number of chromosomes in the normal diploid cell, as well as their size distribution. For example, the human chromosome has 23 pairs of chromosome, 22 somatic pairs and one pair of sex chromosomes. One important aspect of genetic research is correlating changes in the karyotype with changes in the phenotype of the individual. • One important aspect of genetics is correlating changes in karyotype with changes in phenotype. For example, humans that have an extra chromosome 21 have Down's syndrome. Insertions, deletions and changes in chromosome number can be detected by the skilled cytogeneticist, but correlating these with specific phenotypes is difficult. • The first discriminating parameter when developing a karyotype is the size and number of the chromosomes. Although this is useful, it does not provide enough detail to be begin the development of a correlation between structure and function (phenotype). To further distinguish among chromosomes, they are treated with a dye that stains the DNA in a reproducible manner. After staining, some of the regions are lightly stained and others are heavily stained. As described above, the lightly stained regions are called euchromatin, and the dark stained regio

n is called heterochromatin. The current dye of chose is the Giemsa stain, and the resulting pattern is called the G-banding pattern. • C-Value Paradox • In addition to describing the genome of an organism by its number of chromosomes, it is also described by the amount of DNA in a haploid cell. This is usually expressed as the amount of DNA per haploid cell (usually expressed as picograms) or the number of kilobases per haploid cell and is called the C value. One immediate feature of eukaryotic organisms highlights a specific anomaly that was detected early in molecular research. Even though eukaryotic organisms appear to have 2-10 times as many genes as prokaryotes, they have many orders of m

agnitude more DNA in the cell. Furthermore, the amount of DNA per genome is correlated not with the presumed evolutionary complexity of a species. This is stated as the C value paradox: the amount of DNA in the haploid cell of an organism is not related to its evolutionary complexity. (Another important point to keep in mind is that there is no relationship between the number of chromosomes and the presumed evolutionary complexity of an organism.) • C Values of Organisms Used in Genetic Studies

• Species Kilobases/haploid genome E. coli • 4.5 x 103 • Human • 3.0 x 106 • Drosophila • 1.7 x 105 • Maize • 2.0 x 106

• Aribidopsis • 7.0 x 104

• A dramatic example of the range of C values can be seen in the plant kingdom where Arabidopsis represents the low end and lily (1.0 x 10^8 kb/haploid genome) the high end of complexity. In weight terms this is 0.07 picograms per haploid Arabidopsis genome and 100 picograms per haploid lily genome. • Genome - the complete set of chromosomes inherited from a single parent; the complete DNA component of an individual; the definition often excludes organelles

• Packing Ratio of  Chromosome Structure • Packing ratio - the length of DNA divided by the

length into which it is packaged • Shortest human chromosome = 4.6 x 107 bp of DN

A (about 10 times the genome size of  E. coli). • Equivalent to 14,000 µm of extended DNA. • Most condensed state (during mitosis) = the chrom

osome is about 2 µm long. • Packing ratio of 7000 (14,000/2)

• Chromosome Structures Involved in "Packing" DNA • Nucleosome - a bead-like structure achieved by winding DNA aro

und a protein core to produce a "bead-like" structure; packing ratio *6

• 30 nm fiber - a coil of nucleosomes in a helical structure; found in both interphase and mitotic chromosomes; increases the packing ratio to *40

• 700 nm structure and scaffolding - organization of the fibers in loops and scaffolds; gives a final packing ratio of about 1000 in interphase chromosomes and *10,000 in mitotic chromosomes

• Chromatin - the unit of analysis of the chromosome; chromatin reflects the general structure of the chromosome but is not unique to any particular chromosome

• Nucleosome Structure of Chromosomes • Nucleosome - fundamental packaging structure of

DNA that is found in all eukaryotic chromosomes; DNA is wrapped around an octamer of small basic proteins called histones; 146 bp is wrapped around the core and the remaining bases link to the next nucleosome; this structure causes negative supercoiling

• Core histones  in Nucleosomes • H2A, H2B, H3 and H4 • form histone octamer (two of each protein) • these basic proteins have an affinity for DNA • the most abundant proteins associated with DNA • the amino acid sequence of these four histones is co

nserved suggesting a similar function for all

• Core DNA in Nucleosomes • DNA that is associated with the core histones • this length is invariant and is always 146 base pairs • the core DNA forms two loops around the octamer • this permits two regions that are 80 bp apart to be brought in

to close proximity; this may allow coordination between two distal sequences for gene regulation.

• Linker DNA in Nucleosomes • Linker DNA is the DNA that is between each histone octam

er • Can vary in length from 8 to 114 base pairs • Variation is species specific • Variation in linker DNA length has also been associated wit

h the developmental stage of the organism or specific regions of the genome