The Current Factors that Affect Risk Distribution and Structure Around Emerging Ecosystem Markets in China

China is undergoing urbanization at a scale and speed that is unprecedented in human history. Though urbanization has driven Chinese economic growth, it has also caused significant environmental damage. Balancing urban development with environmental protection is one of China’s top challenges over the upcoming decades; as such, Chinese officials have indicated strong interest in utilizing innovative, market-based environmental planning policies such as ecosystem service markets. One such ecosystem service market is mitigation banking, where developers can offset wetland destruction by purchasing “credits” for wetlands that have been restored by mitigation banks in another area. This paper analyzes the feasibility and risk considerations for mitigation banking in mainland China, focusing on regulatory and entrepreneurial risk. My results suggest that the legal and administrative framework for mitigation banking already exists within mainland China; however, regulatory and entrepreneurial risk in the Chinese governmental context are considerably higher than in the United States. For regulators, this is due to several factors: lack of vertical supervision within the Chinese government, the close relationship between real estate and politics, and the general emphasis of GDP growth over environmental protection. For entrepreneurs, increased risk is due to uncertainty around regulations, a weak judicial system, and general corruption. I discuss the implications of these increased risks and suggest solutions.Master of City and Regional Plannin

Book Review: Networks Cities

Post-Mao China has undergone a level of urbanization that is unprecedented in size and speed. Large districts, cities, and even regions have appeared where little used to exist. Chinese planners have generally used traditional, mono-zone strategies with high densities to accommodate this growth. James Brearley and Fang Qun’s bilingual Chinese and English-language book Networks Cities, provides a mixture of essays, master plans, and case studies throughout China that highlight the shortcomings of this traditional planning model and offers a vision on how innovative planning techniques can provide a more sustainable foundation for China’s future growth

Characterization of the Searching Mechanism for a DNA Repair Enzyme.

Genomic DNA is constantly subjected to damaging modifications from reactive metabolites and environmental mutagens. The base excision repair pathway (BER) is responsible for repair of most modifications affecting single bases, collectively referred to as base lesions. Base lesions are the most frequently occurring type of DNA damage, with ~10,000 base lesions formed and repaired by BER in human cells everyday. However, in the context of the human genome, these base lesions are extremely rare, with only one of every 1.2 million nucleotides sustaining damage on any given day. The daunting task of locating single base lesions and initiating the BER pathway is bestowed upon DNA glycosylases, which catalyze removal of a wide variety of damaged nucleobases from DNA. We show that alkyladenine DNA glycosylase (AAG), a human protein that initiates repair of a diverse group of alkylated and deaminated purines, locates damage by a correlated searching mechanism whereby each binding encounter with DNA involves a search of multiple adjacent sites. This search is mediated by electrostatic binding interactions that allow linear diffusion along nonspecific DNA to locate target sites. We show that the N-terminus of AAG, which is dispensable for glycosylase activity, contributes to the correlated search by decreasing dissociation from DNA, possibly by directly contacting DNA. Furthermore, we demonstrate that AAG makes significant excursions from the surface of DNA while diffusing. Such events, referred to as hops, allow reorientation between strands, enabling AAG to search both strands of a DNA duplex in a single binding encounter. Hopping also allows AAG to bypass obstacles, such as tightly-bound proteins and helix-discontinuities. By comparing the behavior of AAG on oligonucleotides containing different lesions, we show that the efficiency of the search for damage depends on the identity of the base lesion. Whereas AAG recognizes the alkylated lesion, 1, N6-ethenoadenine with high efficiency, AAG requires multiple encounters with the oxidative lesion inosine. We infer that a highly redundant search allows multiple encounters with each lesion, ensuring that each lesion is repaired. Collectively, these studies provide insight into the molecular mechanism by which a DNA repair enzyme searches the genome for DNA damage.Ph.D.Chemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/78826/1/mhedgli_1.pd

Structure of the human clamp loader bound to the sliding clamp: a further twist on AAA+ mechanism [preprint]

DNA replication requires the sliding clamp, a ring-shaped protein complex that encircles DNA, where it acts as an essential cofactor for DNA polymerases and other proteins. The sliding clamp needs to be actively opened and installed onto DNA by a clamp loader ATPase of the AAA+ family. The human clamp loader Replication Factor C (RFC) and sliding clamp PCNA are both essential and play critical roles in several diseases. Despite decades of study, no structure of human RFC has been resolved. Here, we report the structure of human RFC bound to PCNA by cryo-EM to an overall resolution of ~3.4 Å. The active sites of RFC are fully bound to ATP analogs, which is expected to induce opening of the sliding clamp. However, we observe the complex in a conformation prior to PCNA opening, with the clamp loader ATPase modules forming an over-twisted spiral that is incapable of binding DNA or hydrolyzing ATP. The autoinhibited conformation observed here has many similarities to a previous yeast RFC:PCNA crystal structure, suggesting that eukaryotic clamp loaders adopt a similar autoinhibited state early on in clamp loading. Our results point to a ‘Limited Change/Induced Fit’ mechanism in which the clamp first opens, followed by DNA binding inducing opening of the loader to release auto-inhibition. The proposed change from an over-twisted to an active conformation reveals a novel regulatory mechanism for AAA+ ATPases. Finally, our structural analysis of disease mutations leads to a mechanistic explanation for the role of RFC in human health

Book Reviews

Book review of the following: Design after Decline: How America Rebuilds Shrinking Cities by Brent Ryan; Good Jobs, Bad Jobs by Arne Kalleberg; Why Good People Can’t Get Jobs by Peter Cappelli; Networks Cities by James Brearley and Fang Qu

PARP2 Promotes Break Induced Replication-Mediated Telomere Fragility in Response to Replication Stress

PARP2 is a DNA-dependent ADP-ribosyl transferase (ARTs) enzyme with Poly(ADP-ribosyl)ation activity that is triggered by DNA breaks. It plays a role in the Base Excision Repair pathway, where it has overlapping functions with PARP1. However, additional roles for PARP2 have emerged in the response of cells to replication stress. In this study, we demonstrate that PARP2 promotes replication stress-induced telomere fragility and prevents telomere loss following chronic induction of oxidative DNA lesions and BLM helicase depletion. Telomere fragility results from the activity of the break-induced replication pathway (BIR). During this process, PARP2 promotes DNA end resection, strand invasion and BIR-dependent mitotic DNA synthesis by orchestrating POLD3 recruitment and activity. Our study has identified a role for PARP2 in the response to replication stress. This finding may lead to the development of therapeutic approaches that target DNA-dependent ART enzymes, particularly in cancer cells with high levels of replication stress

Single-molecule visualization reveals the damage search mechanism for the human NER protein XPC-RAD23B

DNA repair is critical for maintaining genomic integrity. Finding DNA lesions initiates the entire repair process. In human nucleotide excision repair (NER), XPC-RAD23B recognizes DNA lesions and recruits downstream factors. Although previous studies revealed the molecular features of damage identification by the yeast orthologs Rad4-Rad23, the dynamic mechanisms by which human XPC-RAD23B recognizes DNA defects have remained elusive. Here, we directly visualized the motion of XPC-RAD23B on undamaged and lesion-containing DNA using high-throughput single-molecule imaging. We observed three types of one-dimensional motion of XPC-RAD23B along DNA: diffusive, immobile and constrained. We found that consecutive AT-tracks led to increase in proteins with constrained motion. The diffusion coefficient dramatically increased according to ionic strength, suggesting that XPC-RAD23B diffuses along DNA via hopping, allowing XPC-RAD23B to bypass protein obstacles during the search for DNA damage. We also examined how XPC-RAD23B identifies cyclobutane pyrimidine dimers (CPDs) during diffusion. XPC-RAD23B makes futile attempts to bind to CPDs, consistent with low CPD recognition efficiency. Moreover, XPC-RAD23B binds CPDs in biphasic states, stable for lesion recognition and transient for lesion interrogation. Taken together, our results provide new insight into how XPC-RAD23B searches for DNA lesions in billions of base pairs in human genome

Searching for DNA Lesions: Structural Evidence for Lower- and Higher-Affinity DNA Binding Conformations of Human Alkyladenine DNA Glycosylase

To efficiently repair DNA, human alkyladenine DNA glycosylase (AAG) must search the million-fold excess of unmodified DNA bases to find a handful of DNA lesions. Such a search can be facilitated by the ability of glycosylases, like AAG, to interact with DNA using two affinities: a lower-affinity interaction in a searching process and a higher-affinity interaction for catalytic repair. Here, we present crystal structures of AAG trapped in two DNA-bound states. The lower-affinity depiction allows us to investigate, for the first time, the conformation of this protein in the absence of a tightly bound DNA adduct. We find that active site residues of AAG involved in binding lesion bases are in a disordered state. Furthermore, two loops that contribute significantly to the positive electrostatic surface of AAG are disordered. Additionally, a higher-affinity state of AAG captured here provides a fortuitous snapshot of how this enzyme interacts with a DNA adduct that resembles a one-base loop.National Institutes of Health (U.S.) (grant no. P30-ES002109)National Institutes of Health (U.S.) (grant no. GM65337)National Institutes of Health (U.S.) (grant no. GM65337-03S2)National Institutes of Health (U.S.) (grant no. CA055042)National Institutes of Health (U.S.) (grant no. CA092584)Repligen Corporation (KIICR Graduate Fellowship

ROS1 5-methylcytosine DNA glycosylase is a slow-turnover catalyst that initiates DNA demethylation in a distributive fashion

Arabidopsis ROS1 belongs to a family of plant 5-methycytosine DNA glycosylases that initiate DNA demethylation through base excision. ROS1 displays the remarkable capacity to excise 5-meC, and to a lesser extent T, while retaining the ability to discriminate effectively against C and U. We found that replacement of the C5-methyl group by halogen substituents greatly decreased excision of the target base. Furthermore, 5-meC was excised more efficiently from mismatches, whereas excision of T only occurred when mispaired with G. These results suggest that ROS1 specificity arises by a combination of selective recognition at the active site and thermodynamic stability of the target base. We also found that ROS1 is a low-turnover catalyst because it binds tightly to the abasic site left after 5-meC removal. This binding leads to a highly distributive behaviour of the enzyme on DNA substrates containing multiple 5-meC residues, and may help to avoid generation of double-strand breaks during processing of bimethylated CG dinucleotides. We conclude that the biochemical properties of ROS1 are consistent with its proposed role in protecting the plant genome from excess methylation

The Influence of Transcription Factor Competition on the Relationship between Occupancy and Affinity

Transcription factors (TFs) are proteins that bind to specific sites on the DNA and regulate gene activity. Identifying where TF molecules bind and how much time they spend on their target sites is key to understanding transcriptional regulation. It is usually assumed that the free energy of binding of a TF to the DNA (the affinity of the site) is highly correlated to the amount of time the TF remains bound (the occupancy of the site). However, knowing the binding energy is not sufficient to infer actual binding site occupancy. This mismatch between the occupancy predicted by the affinity and the observed occupancy may be caused by various factors, such as TF abundance, competition between TFs or the arrangement of the sites on the DNA. We investigated the relationship between the affinity of a TF for a set of binding sites and their occupancy. In particular, we considered the case of the transcription factor lac repressor (lacI) in E.coli, and performed stochastic simulations of the TF dynamics on the DNA for various combinations of lacI abundance and competing TFs that contribute to macromolecular crowding. We also investigated the relationship of site occupancy and the information content of position weight matrices (PWMs) used to represent binding sites. Our results showed that for medium and high affinity sites, TF competition does not play a significant role for genomic occupancy except in cases when the abundance of the TF is significantly increased, or when the PWM displays relatively low information content. Nevertheless, for medium and low affinity sites, an increase in TF abundance (for both cognate and non-cognate molecules) leads to an increase in occupancy at several sites. © 2013 Zabet et al